In situ heat induced antigen recovery and staining apparatus and method

ABSTRACT

An automated microscope slide staining system and staining apparatus and method that features a plurality of individually operable miniaturized pressurizable reaction compartments or a pressurizable common chamber for individually and independently processing a plurality of microscope slides. The apparatus preferably features independently movable slide support elements each having an individually operable heating element.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 15/050,132,filed Feb. 22, 2016; which is a continuation of U.S. Ser. No.13/943,510, filed Jul. 16, 2013, now U.S. Pat. No. 9,267,868; which is acontinuation of U.S. Ser. No. 12/550,296, filed Aug. 28, 2009, now U.S.Pat. No. 8,486,335; which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/190,503, filed Aug. 29, 2008; each of which ishereby expressly incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

BACKGROUND

The present invention is related to the field of treating samples onmicroscope slides or other analytical substrates, and more specificallyto the field of heat induced antigen recovery and staining of suchsamples.

In anatomical pathology labs (e.g., histology, cytology) it is knownthat certain immunohistochemical procedures, herein known as IHC assays,are performed on biological specimens including, for example,formalin-fixed paraffin-embedded tissues and cell preps. Also used inthe art are several IHC antibodies (abs) like Estrogen receptor abs,Progesterone receptor abs, Proliferation abs like Ki-67, which requirethe use of high temperature unmasking techniques, (i.e., antigenretrieval, high temperature epitope recovery, and antigen unmasking),prior to application of the antibody for labeling cell structures(antigens).

There are several procedures known in the art for the “unmasking” ofantigens that have been rendered “hidden” by formalin fixation.Procedures known in the art include treating the biological specimen inaqueous solutions (e.g., water) that may include buffers (e.g., citrate,EDTA, urea, etc.), along with detergents or surfactants (e.g., Brij 35,Tween, SDS, NP-40 and Igepal). These known formulations are heated totemperatures from around 60° C. to about 120° C. These heatedformulations are in contact with the biological specimen for variousamounts of time (e.g., about 10 minutes to about 90 minutes) therebycausing the “masked” antigen to become “unmasked” so the antibodies usedin the IHC assays can attach to their corresponding antigens which areassociated with the biological specimen.

Types of apparatuses that are known and used to perform the heating ofthe antigen retrieval solutions and the biological specimen includewaterbaths, steamers, pressure cookers, autoclaves, microwave ovens andconvection ovens. Since water boils at 100° C. at normal atmosphericpressure, antigen retrieval solutions even with other chemicals presenthave only been able to reach temperature from about 98° C. to 100° C.before evaporative heat loss inhibits the solution from reaching highertemperatures. Pressure cookers and autoclaves overcome this by allowingfor pressurization of the solutions so higher temperatures can beachieved without evaporation of the heated fluid. Since there areantibodies that require the antigen retrieval solution be attemperatures exceeding 100° C., many laboratories must use pressurecookers to heat the biological specimen with its antigen retrievalsolution to attain temperatures up to 120° C., without which the antigenwould not be “unmasked” preventing the antibody from binding to theantigen.

There remains a need for an apparatus able to produce high temperatureand pressure conditions for single slides being subjected toindividualized antigen retrieval conditions without relying on clumsyand unwieldy devices such as pressure cookers and autoclaves.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a microscope slide staining system of theinvention.

FIG. 2 is a front cross-sectional view of a staining apparatus of amicroscope slide staining system of the present invention.

FIG. 3A is a perspective view of the staining apparatus of FIG. 2.

FIG. 3B is a perspective view of a microscope slide staining system ofthe present invention having four staining apparatuses such as theapparatus of FIG. 3A.

FIG. 4 is a top plan view of the staining apparatus of FIG. 3A.

FIG. 5 is a perspective view of the staining apparatus of FIG. 3A shownas having three slide support elements ejected from the inner space ofthe staining apparatus.

FIG. 6 is a top plan view of the staining apparatus of FIG. 5.

FIG. 7 is a cross-sectional side view of a set of reaction components(e.g., reaction compartment, slide support element, and reagent packsupport device) in a staining apparatus of the present invention beforethe reagent pack has been inserted into the reagent pack support device,and before a microscope slide has been disposed on the slide supportelement. The walls of the staining apparatus are not shown forsimplification.

FIG. 8A is a cross-sectional side view of the reaction components ofFIG. 7 in operation in a reagent dispensing phase.

FIG. 8B is a transverse cross-sectional view of the reaction componentsof FIG. 8A.

FIG. 9A is a cross-sectional side view of the reaction components ofFIG. 7 and FIG. 8A in a reagent drainage phase.

FIG. 9B is a transverse cross-sectional view of the reaction componentsof FIG. 9A.

FIG. 10A is a cross-sectional side view of the reaction components ofFIG. 9A in a rinse buffer dispensing phase.

FIG. 10B is a transverse cross-sectional view of the reaction componentsof FIG. 10A.

FIG. 11A is a cross-sectional side view of the reaction components ofFIG. 10A in a rinse buffer drainage phase.

FIG. 11B is a transverse cross-sectional view of the reaction componentsof FIG. 11A.

FIG. 12 is a cross-sectional view of the reaction components of FIGS.7-11B after the reagent pack is completely used and the microscope slideis removed from the slide support element.

FIG. 13 is an enlarged version of FIG. 8A.

FIG. 14 is an enlarged version of FIG. 10A.

FIG. 15A is a top plan view of the reaction compartment and slidesupport element of FIG. 13 which shows a clockwise air mixing step.

FIG. 15B is a transverse cross-sectional view of the air ports of theslide support element of FIG. 15A.

FIG. 16A is a top view of the reaction compartment and slide supportelement of FIG. 13 which shows a counter-clockwise air mixing step.

FIG. 16B is a transverse cross-sectional view of the air ports of theslide support element of FIG. 16A.

FIG. 17 is a view of the microscope slide and detached components of theheating element of the slide support element of FIG. 12.

FIG. 18A is a top plan view of a slide support element with themicroscope slide and heating element detached to show air flow throughthe air cooling ducts which are used to enhance a rapid cooling of theheating element.

FIG. 18B is a transverse cross-sectional view through the air coolingducts of the slide support element of FIG. 18A.

FIG. 19A is a cross-sectional side view of the reaction components ofFIG. 18A.

FIG. 19B is a transverse cross-sectional view through the air coolingducts of the slide support element of FIG. 19A.

FIG. 20 is a view of the microscope slide and detached components of theheating element of the slide support element of FIG. 12.

FIG. 21A is a top plan view of a slide support element with themicroscope slide and heating element detached to show air flow throughthe air cooling ducts which are used to rapidly cool the heatingelement.

FIG. 21B is a transverse cross-sectional view through the air coolingducts of the slide support element of 21A.

FIG. 22A is a cross-sectional side view of the reaction components ofFIG. 18A.

FIG. 22B is a transverse cross-sectional view through the air coolingducts of the slide support element of FIG. 22A.

FIG. 23 is a cross-sectional side view of an alternate embodiment of thereaction components, particularly the slide support element, of thepresent invention.

FIG. 24 is a cross-sectional side view of the reaction components ofFIG. 23 in an alternate processing configuration wherein a reagent ofthe reagent pack is applied to the microscope slide outside of thereaction compartment.

FIG. 25 is a cross-sectional side view of the reaction components ofFIG. 23 in another alternate processing configuration wherein a reagentfrom a remote source is applied to the microscope slide outside of thereaction compartment.

FIG. 26 is a cross-sectional side view of the reaction components ofFIG. 23 in an alternate processing configuration.

FIG. 27 is a cross-sectional side view of the reaction components ofFIG. 23 in an alternate processing configuration.

FIG. 28 is an enlarged fragmented cross-sectional side view of thereaction components of FIG. 23 in an alternate processing configuration.

FIGS. 29A-29F are cross-sectional side views of an embodiment of theinvention wherein the slide support element is able to move into and outof the staining apparatus and reaction compartment, and the reactioncompartment is able to move backwardly to enable application of thereagents directly onto the microscope slide on the slide supportelement.

FIGS. 30A-30F are cross-sectional side views of an embodiment of theinvention wherein the slide support element is able to move to variablepositions within the reaction compartment such that the pressurizationwithin the reaction compartment is able to occur via compression of thehead space (“in-situ” pressurization) of the reaction compartment by theslide support element.

FIGS. 31A-31F are cross-sectional side views of an embodiment of theinvention which are similar to those of FIGS. 29A-29F, except thereaction compartment has an upper window through which reagents can beapplied to the microscope slide without requiring movement of thereaction compartment backwardly. The reaction compartment can be rotated180° (for example) to enclose the microscope slide within apressurizable portion of the reaction compartment.

FIG. 32 is a perspective view of a reaction compartment having a window,such as is used in the embodiment of FIGS. 31A-31F.

FIGS. 33A-33H are cross-sectional side views of an embodiment of theinvention combining the “window” elements of FIGS. 30A-30F and the“in-situ” pressurization elements of FIGS. 31A-31F.

FIG. 34 is a top plan view of an apparatus of the invention similar tothe apparatus of FIG. 4 except additionally having an X-Y-Z positioningapparatus comprising a dispenser head and a rotary reagent carouselcomprising a plurality of reagent vials for dispensing reagents onto themicroscope slides.

FIG. 35 is a top plan view of an apparatus similar to the apparatus ofFIG. 34 except further having a separate pressurizable common chamberisolated from an application chamber in which reagents are applied tothe microscope slides.

FIG. 36 is a top plan view of an apparatus similar to the stainingapparatus of FIG. 4 except in place of separately pressurizable reactioncompartments, the apparatus comprises a pressurizable common chamberinto which the slide support elements can be withdrawn and treated undera common pressure level.

FIGS. 37A-37F shows a gap coating mechanism which causes a reagent to bespread over the biological specimen on the microscope during operationof the present invention. FIGS. 37A, 37B and 37C are cross-sectionalviews.

FIGS. 37D-37F are top views.

FIGS. 38A-38B shows top plan views of an alternate gap coater of theinvention.

FIGS. 39A-39B are top plan views of alternate embodiments of the gapcoater of the invention.

FIG. 40 is a top view of a reagent pack of the present invention.

FIG. 41A is a cross-sectional view taken through line 41A/41B of FIG. 40which shows reagent containers as blisters or bubbles.

FIG. 41B is a cross-sectional view taken through line 41A/41B of FIG. 40which shows the reagent containers as vials.

FIG. 42 is a top plan view of an attachable/detachable module of thereagent pack of FIG. 40 having a single reagent container thereon.

FIG. 43A-43B is a cross sectional side view of a slide supportembodiment wherein the slide support element has a beveled seal forsealing with a front wall of the staining apparatus.

DETAILED DESCRIPTION OF THE INVENTION

Contemplated herein is an automated microscope slide staining systemthat features an apparatus comprising a plurality of independentlymovable and operable slide support elements for individually andindependently processing and pressurizing a plurality of individualmicroscope slides. Where used herein the term “microscope slide” isintended to refer to conventional microscope slides as well as othermicroscopy analytical devices which are used as vessels, substrates, orsupport structures for supporting biological and biochemical specimensfor testing, processing and/or analysis, and which are sized and shapedto fit on a support element as described and contemplated herein. Thusthe term “microscope slide” includes, but is not limited to, devicessuch as biochips, vials, flasks, microtiter plates, test tubes, petridishes, and microarray plates, as well as standard glass or plasticmicroscope slides. Preferably, the apparatus of the present invention isused as an automated in-situ antigen recovery and staining apparatus andpreferably features independently movable slide support elements, eachof which has an individually heatable heating plate or elementassociated therewith. Each slide support element preferably supports asingle microscope slide. Each slide support element with the microscopeslide thereon is, in a preferred embodiment, enclosable within its ownindividually and independently pressurizable reaction compartment and/orcomprises a portion thereof. In one treatment step, for example, asolution such as an antigen retrieval solution is disposed on themicroscope slide and the heating plate or element heats the slide andthe antigen retrieval solution thereon to temperatures of, for example,120° C. to 160° C. by regulating the pressure within the individualreaction compartment (or pressurizable common chamber of the stainingapparatus as explained below) thereby increasing the temperature thatthe solution can attain. In one embodiment each reaction compartment hasits own individual pressure regulator, device, or switch to regulatepressure within the reaction compartment but more preferably pressure isregulated by modulating heat and pressure within the reactioncompartment. Pressures exceeding 1 atm (i.e., exceeding 14.7 psi, 0 psigor 101.325 kPa) or below 1 atm can be created and maintained in thereaction compartment and the biological specimen on the microscope slideis exposed to this pressure level. The reaction compartment can hold,for example, 0.1 ml to 100 ml of antigen retrieval solution.

Where used herein the term “biological specimen” includes, but is notlimited to, unprocessed specimens, processed specimens, paraffinembedded tissue, whole mounts, frozen sections, cell preps, cellsuspensions, touch preps, thin preps, cytospins, and other biologicalmaterials or molecules including blood, urine, cerebrospinal fluids,pleural fluids, ascites fluids, biopsy materials, fine needle aspirates,pap smears, swabbed cells or tissues, microbiological preps includingbacteria, viruses, parasites, protozoans, biochemicals including, butnot limited to proteins, DNA, RNA, carbohydrates, lipids, ELISA reagentsand analytes, synthetic macromolecules, phospholipids, supportstructures of biological molecules (e.g., metals, beads, plastics,polymers, glass), or any other materials attached to a biologicaltesting substrate for processing, examination, or observation.

Each microscope slide at some point during treatment is treated with aliquid solution or reagent (generally referred to herein as “reagents”or “reagent elements” and including, but not limited to, antigenretrieval reagents, molecular RNA and DNA probes, citrate buffer, EDTA,TRIS, PBS, with or without surfactants or detergents like SDS, Tween,Brij, ionic and non-ionic detergents, and silicone additives, rinsebuffers, immunoreagents, immunohistochemical reagents, biologicalstains, histochemical reagents, counterstains, in-situ hybridizationreagents, chromogens, PCR reagents, monoclonal antibodies, polyclonalantibodies, coverslipping reagents, silicone oils, mineral oils,detection reagents and processing reagents, liquid reagents,reconstituted dry reagents, biological reagents and aqueous andnon-aqueous reagents, and deparaffinizing compositions of water with oneor more silicone surfactants or silicone additives). Because of theability to pressurize and regulate pressure within the reactioncompartment, and the ability to individually heat each slide, each slidecan be heated to temperatures that could not be obtained without theelevated pressurized environment of the enclosed reaction compartment(or pressurizable common chamber). For example, since the vapor producedby the reagent is contained in the reaction compartment (or is releasedin a regulated manner), the pressure in the reaction compartment can beregulated to produce a reaction temperature required by the user.Pressures (“negative pressure”, i.e., vacuums) below 1 atm (i.e., below14.7 psi, 0 psig or 101.325 kPa) may also be created and maintainedwithin the reaction compartment. For example, vacuum pressures of from100 kPa to 10 kPa to 1 kPa to 100 Pa to 10 Pa to 1 Pa to 0.1 Pa may beformed and held in the reaction compartment.

In preferred embodiments, each reaction compartment and microscope slidecan be heated separately and independently from the other reactioncompartments and microscope slides by a conductive heating element (orheating plate) underneath or otherwise adjacent to the microscope slide(e.g., wherein the heating element is in a sidewall of the reactioncompartment or in a cavity). In one embodiment in an enclosed reactioncompartment, the microscope slide therein has an antigen retrievalsolution deposited onto the microscope slide before or after beingplaced in the reaction compartment. The slide is then heated, in apreferred embodiment, to a temperature of about 100° C.-300° C. andunder a pressure from 0.1 psig (102.015 kPa) to, for example, 350 psig(2515 kPa). In one embodiment the containment of the pressure isproportional to the temperature of the antigen retrieval solution, suchthat the regulation of both the temperature of the heating element ofthe reaction compartment and the regulation of the pressure generated bythe solution on the slide can be adjusted during the automated stainingprocedure.

In one example, the heating element could heat the slide to 120° C. orgreater and the pressure in the reaction compartment could be 16 psig(30.7 psi) wherein the solution on the microscope slide in contact withthe biological specimen would be about 130° C., for example. It would beapparent to one of ordinary skill in the art of pressure regulatedvessels that the temperature attained by the antigen retrieval solutionwould be dependent on the regulation and containment of either thepressure generated or the temperature of the heating element or both. Ifregulation of the temperature of the solution is to be at leastpartially determined by the pressure level in the reaction compartment,the heating plate can be set at 130° C. (for example) and the pressurerelief valve could be set to a level to maintain a pressure of 19 psig(232.4 kPa) within the reaction compartment, for example. Thus, thetemperature of the antigen retrieval solution would not substantiallyexceed 130° C. and would remain in the range of 120° C.-130° C.

If regulation of the temperature of the solution on the microscope slideis desired to be regulated by the temperature of the heating element,then the heating plate can be regulated to heat the slide to a desiredtemperature. Once the desired pressure within the reaction compartmenthas been reached, the temperature of the heating element is adjusted tokeep the desired pressure within the desired limits. The reactioncompartment under some conditions does not necessarily require apressure regulator since the pressure in the reaction compartment may bedetermined solely by the temperature level of the heating element. Insome embodiments it would be advantageous to have a regulator to relievepressure if the pressure exceeds desired levels or to have a pressureregulator which would cause the heating element to be turned on and offdepending on the desired pressure level.

Since “boiling” of the solution or reagent on the slide is suppressed bythe containment of the pressure, the antigen recovery buffer or otherreagent on the microscope slide may appear not to be boiling(“bubbling”) even though it has actually reached a temperature at orabove 100° C. Elimination or reduction of vapor loss due to boiling isadvantageous because it removes the necessity of adding additionalbuffer during processing (such as is necessary when using certain otherapparatuses known in the art, e.g., as shown in U.S. Pat. Nos.5,654,200; 5,595,707; 6,296,809; 6,352,861; 6,582,962; 6,096,271;6,180,061; 6183,693; 6,541,261; or 6,783,733). This removal of thenecessity to add reagent during treatment occurs even when only smallamounts of buffers or reagents are initially used (e.g., 500 μ1-4 ml)and treatment times may be extended up to 60 minutes at hightemperatures (e.g., over 100° C., e.g., 120° C.-160° C.). Loss ofreagent volume during heating in the present invention is thus minimaldue to containment of vapors generated. Another important advantage tominimization of boiling at high temperatures is that the biologicalspecimen on the slide is not subjected to extreme agitation from bubbleformation which could cause the biological specimen to detach from theglass slide or be otherwise damaged. Moreover, the controlledpressurized micro-environment in the reaction compartment of the presentinvention is very efficient because the amount of buffer that is used isminimal and the amount of time needed to heat to high heat conditions(e.g., 120° C.-160° C.) is also minimal (e.g., 5 minutes).

Commercial pressure cookers which are currently available for use inantigen retrieval procedures are bulky and require a greater amount ofbuffer or reagent and time to complete the high temperature antigenretrieval process and furthermore must be used to treat many slides inthe same container. The typical pressure cooker treatment cycle fromstart time to the last step (rinse) typically lasts 45-60 minutes. Onlya few different buffers can be heated at the same time, (on the order of5-6 separate slide treatment containers) within a pressure cooker's mainreaction compartment. Moreover, each separate slide container in aconventional commercial pressure cooker requires significant volumes ofantigen retrieval solution (e.g., 45-50 mls per container). As opposedto the pressure cookers which are used in the field of antigenretrieval, the apparatus and method of the present invention may use thevapor pressure generated by the reagent on the slide itself to producean elevated pressure in the individual reaction compartment.Conventional pressure cookers, to the contrary, rely on a separateliquid present within the bottom of the vessel to produce the vapornecessary to cause increased pressure within the vessel for inducingantigen retrieval on the slides therein. This method requires theadditional step of heating the separate liquid to an elevatedtemperature before the process of heating the slide and the reagentthereon can begin.

Each of the individual reaction compartments of the apparatus of thepresent invention, to the contrary, utilize relatively small quantitiesof antigen retrieval buffer (e.g., 0.5-5 ml per slide) and heat upquickly and cool quickly due to the small amounts of liquid and area tobe heated and cooled. Even a volume of 0.1-1 ml per slide can be usedwith the present invention and the typical time from start to finishusing the present invention may be just 20 minutes, for example.

In a preferred embodiment of the invention, to prevent small amounts ofliquid reagents (e.g., including, but not limited to antigen retrievalreagents, RNA and DNA probes, citrate buffer, EDTA, TRIS, PBS, with orwithout surfactants or detergents like SDS, Tween, Brij, ionic and nonionic detergents, and silicone additives, rinse buffers,immunohistochemical reagents, histochemical reagents, in-situhybridization reagents, PCR reagents, coverslipping reagents, siliconeoils, mineral oils, detection reagents and processing reagents, liquidreagents, reconstituted dry reagents, biological reagents and aqueousand non-aqueous reagents, and deparaffinizing compositions of water withone or more silicone surfactants or silicone additives, or other reagentelements described herein) from being reduced in volume by theconversion from a liquid phase to a gaseous phase, and loss thereof,during heating (as occurs in other commercially available systems), thereaction compartment of the staining apparatus of the present invention,when closed, can be pre-pressurized, individually, prior to the heatingof the slide and reagent. This pre-pressurization from a separatepressurization source, (i.e., rather than solely from the vapor pressureproduced by the heated liquid in the reaction compartment), cansignificantly reduce the amount of loss of the gaseous phase(evaporation) of the small amounts of liquid reagents (e.g., 100 μl-5ml) under high temperature conditions (e.g., 100° C.-160° C.) forextended heating times (e.g., 10-60 minutes) of the present invention,thereby eliminating the requirement of adding additional reagent afterthe treatment process has begun (i.e., after the reaction compartment orslide support element is isolated within the staining apparatus). Forexample, preferably, 0.1-4 milliliters of the reagent element (e.g.,antigen retrieval reagent) is placed on the slide, the reactioncompartment is then pre-pressurized and then the heating element beginsto heat the reagent. The pre-pressurization of the reaction compartment,followed by heating of the reagent, produces an environment for thereagent to reach temperatures exceeding 100° C., for example up to 160°C., with minimal reagent loss due to gas phase formation (evaporation).

It is apparent that with the present invention particular temperaturesand pressures can alternatively be established at any desired level forany treatment protocol known in the art of staining biologicalspecimens. Super high temperature conditions can also be achieved usingthe present invention. These super high heating conditions can reach andexceed, for example, 350° C. and 300 psig (2170 kPa) due topressurization, pre-pressurization, and the particular construction ofthe reaction compartment (described in further detail below). Theindividual pre-pressurizable reaction compartments of the presentinvention can be adapted to hold any type of vessel or substrate knownin the art for containing a biological specimen for testing as describedelsewhere herein.

In a preferred embodiment, the reaction compartment can bepre-pressurized and remain pressurized even under very high pressures ofover 300 psig (2170 kPa) to produce very high temperatures exceeding300° C. for use in special procedures that require such very hightemperature conditions. In alternate embodiments, the reactioncompartment can generate and sustain temperatures and pressures, forhigh heat conditions, in the range of 100° C. to 160° C. to 200° C. to250° C. to 300° C., for example. Preferably, a pressure of at least 15psig (204.8 kPa) is maintained within the reaction compartment duringheating.

As described elsewhere herein, this heat can be generated by aconductive heating element positioned on or in the slide support elementbeneath the microscope slide, a conductive heating element in thereaction compartment wall, other types of heating devices in locationsadjacent to the reagents being heated, microwaves passed into thereaction compartment to heat the regents, and/or magnetic induction forexample. These types of heating devices can all be incorporatedseparately or together with the systems described herein for theregulation of pressure.

The regulation of pressure within the reaction compartment (orpressurizable common chamber), either by pre-pressurization from anextended source, or by pressure produced by evaporation of the heatedreagent, or other means such as in situ pressurization described herein,is an important component of the invention.

A preferred embodiment the present invention eliminates the use of asingle large container (e.g., a pressure cooker) to treat one or aplurality of slides under pressure. Each individually operable reactioncompartment of a staining apparatus of the present apparatus can treatat least one individual microscope slide disposed therein with one ormore individually applied reagents at an individualized temperature andpressure without relying on or affecting any of the other plurality ofmicroscope slides in their respective reaction compartments in the sameapparatus, i.e., each pressurizable reaction compartment can operateindependently of each other pressurizable reaction compartment. Anadvantage of this embodiment of the invention is in its ability to treatevery slide in the instrument separately and independently at anindividualized temperature and pressure within a dedicated reactioncompartment thereby increasing efficiency in the production andprocessing of specimens and providing a constant workflow advantage.Using this embodiment of the invention, a technician can separatelybegin a test of a slide utilizing any protocol at any temperature orpressure without affecting or stopping the other reaction compartmentseven when those other reaction compartments are already in use.

As described above, the temperature of the reagent on the microscopeslide on the slide support element can be maintained by regulating thetemperature of the heating element or by regulating the pressure towhich the microscope slide is exposed or by both in combination. In oneembodiment, for example, the heating element can be set to reach 125°C., the maintenance pressure can be set to 23 psig (259.9 kPa), and thereaction compartment can be pre-pressurized to 23 psig (259.9 kPa), andthe slide can be heated such that the reagent on the microscope slidereaches a temperature of 125° C. for 10 minutes, and is then cooled forfurther processing. In a preferred embodiment, the pre-pressurizedconditions may be attained before the microscope slide is heated sothat, in this embodiment, the pressure in the reaction compartment isnot produced by the vaporization of the liquid reagent contained in thereaction compartment, but rather by a separate pressurization method,system or device. The reaction compartment preferably holds a singlemicroscope slide but can be adapted to hold two or more microscopeslides. In the preferred embodiment, an individual reaction compartmentis pre-pressurizable and is constructed to contain only a singlemicroscope slide.

Without wishing to be held to theory, the pre-pressurization process,when using reagents (including any reagents described elsewhere herein)features conditions to minimize evaporative loss of reagents and oraqueous phase (water) or oil phase (oil) of reagents during heatingand/or ambient temperature staining conditions. A further aspect of theembodiment featuring the independently pre-pressurized reactioncompartments is that during the reaction process, pressure within thereaction compartment causes the reagents to come in close physicalcontact with the biological specimen by being “pressed” against thebiological specimen wherein the physical contact between them isincreased due to the pressure exerted on the reagent and thereby of thereagent upon the biological specimen.

This pressurized force of the reagent upon the biological specimen onthe microscope slide helps to decrease the time of treatment by thereagents due to very efficient contact of the reagents with thebiological specimen. Specimens may have their processing timessignificantly reduced due to superior staining caused by the reagentsbeing physically “pressed” against the biological specimen, thusenhancing intimate contact with the biological specimen.

Polymerase Chain Reaction (PCR), including tissue PCR, is dependant onthe retention of the water levels in the reagents during processing.Specific water concentrations, pH conditions, and temperatures must bestrictly met in order for the PCR reaction to be successful. Thepressurized conditions of the reaction compartment of the presentinvention are ideal for these conditions (low evaporation) to be metduring staining. This low evaporation, due to an individuallypressurized micro-environment (the individual reaction compartment) isideal for PCR reactions on glass microscope slides, plastic microscopeslides, vessels, tubes, micro arrays, micro titer plates, plates, or anyother vessel used for the containment of biological specimens. Thispressurization can also be used at ambient temperature as well (e.g.,25° C.).

In one embodiment of the apparatus, the pre-pressurizable reactioncompartments are sized to hold only one microscope slide, while in analternate embodiment, the reaction compartment can hold severalmicroscope slides e.g., two, three, four, or more and can bepre-pressurized to decrease processing time and reduce evaporation orreagent loss.

The heating of the reagent on the microscope slide can be done bypre-pressurizing the reaction compartment with heated (below 100° C.) orsuperheated (above 100° C.) air (or gas) that would maintain therequired temperature for the treatment protocol or would at leastpre-heat the reaction compartment prior to the heating element reachingheating temperature or being turned on to heat, and maintain the heatingof the reagent on the microscope slide. As noted above, in aparticularly preferred embodiment of the invention, one or more of thereaction compartments of the staining apparatus is pre-pressurized afterthe microscope slide or slides are enclosed therein. Thepre-pressurization of the reaction compartment may occur before, during,or after the heating element is actuated to heat the microscope slideand reagent thereon.

In another embodiment of the invention in which the apparatus comprisesa pressurizable common chamber for pressurization without separatepressurizable reaction compartments (e.g., see FIGS. 35-36 below), aplurality of microscope slides together in the pressurizable commonchamber may be pre-pressurized and heated thereby eliminating the needto add additional reagent to each microscope slide during the antigenretrieval process. For example, the plurality of microscope slides inthe apparatuses shown in U.S. Pat. Nos. U.S. 5,654,200; 5,595,707;6,296,809; 6,352,861; 6,582,962; 6,096,271; 6,180,061; 6183,693;6,541,261; or 6,783,733 may be enclosed within a pressurizable commonchamber and pre-pressurized before, during, or after the heating stepbegins. In this embodiment, a plurality of microscope slides onindependently movable slide support elements are enclosed within apressurizable common chamber, reagent is applied to the microscopeslides (before or after enclosure within the pressurizable commonchamber), the pressurizable common chamber is pressurized to a levelabove atmospheric pressure, and the microscope slides are heated so thetemperature of the reagent on the microscope slide exceeds 85° C. andmore preferably exceeds 100° C. Further, the reagent could be applied tothe microscope slides after the pressurizable common chamber ispressurized.

The same steps as above could be followed in an alternate embodimentabsent inclusion of a heating process. The result of the process withoutheating is reduced evaporation or vaporization of the reagent from theslide while reagent is reacting with the specimen or sample on the slideand an increase in the physical interaction thereof, due to increasedpressure of the reagent with the specimen or sample on the slide.

In a preferred embodiment wherein the apparatus comprises separatepressurizable reaction compartments, each microscope slide on eachseparate slide support element is processed within its own individualreaction compartment that can be individually pressurized. Each reactioncompartment is operable separately from each other reaction compartment.Together they comprise an automated slide staining apparatus able toprocess a plurality of microscope slides simultaneously, if desired, yetindependently. Each reaction compartment (and slide support element) isfunctionally operably independent (i.e., non-interdependent) from eachother reaction compartment. The independent operability of each reactioncompartment (and slide support element) is due to each reactioncompartment having separate operational mechanisms, including but notlimited to, individually moving slide support elements, individuallymoving reagent dispensing packs and/or reagent dispensing devices, andindividually movable or stationary ports and dispensers for rinses,pressure, vacuum and waste disposal. Preferably each single individualprocessing device corresponding and dedicated to any of the reactioncompartments is independent at any time of the operation of thededicated processing components of another reaction compartment whetherit is in operation or not, including, preferably, micro-processingprograms unique to each reaction compartment. All processing components(e.g., including, but not limited to, reagent dispensers, rinse ports,vacuum ports, pressure ports, waste ports, mixing ports, slide supportelements, reaction compartments, air cooling ducts, and liquid coolingducts) can be individually and independently moveable and/or usable. Theexception to this, in an embodiment of the apparatus, is one or more“X-Y-Z” positioning devices discussed elsewhere herein (e.g., FIGS. 34and 35).

The apparatus of the present invention preferably comprises amicroprocessor which utilizes an operating system that can havemultiple, individually, and/or simultaneously running processingprograms, partially or completely specific to each individual reactioncompartment and/or slide support element. This would enable a simpleapproach to programming by eliminating the need for the microprocessorto have one operating program to determine and evaluate the status ofall processing steps as in current slide staining instruments (e.g., asshown in U.S. Pat. Nos. 5,439,649, 5,595,707, 5,758,033, 5,839,091,6,296,809, 6,352,861 and 6,783,733). In such staining instruments knownin the prior art, microprocessors have a processing program which is“aware” of all the steps for each microscope slide in the stainingprocess and which determines the correct time to activate a commonprocessing device for a particular slide's use (i.e. reagent dispenser,rinses, air applications, etc.) This “thinking and reacting” approach tothe microprocessor's involvement in processing a plurality of microscopeslides is inefficient. A lag time is produced when all the microscopeslides are under the control of one program. This inefficient use oftime causes increased time for processing just because of therequirement of the microprocessor to determine the next step for eachmicroscope slide and determine any conflicts with two or more microscopeslides needing to be processed by a common device at the same time. Thistype of micro-processing delays the completion of the processing of amicroscope slide that would need a processing device at the same time asanother microscope slide or multiple microscope slides.

Some staining instruments known in the art feature a “STAT RUN” option.With this type of processing, the user has already started a stainingrun and has decided that one or more additional microscope slides needto be placed on the instrument and processed because the processing ofthe “additional microscope slides” is more urgent. The user can put the“original” microscope slides on a lesser priority setting. The “newmicroscope slides” can then be placed on the instrument and wouldreceive the priority use of the “new microscope slides” of all theprocessing devices (e.g., reagent dispensers). In between the prioritystaining protocol, the processing devices can then be used to treat the“original” or “non-stat” microscope slides that were on theindependently operable instrument initially. The requirement for thistype of interrupted processing is eliminated due to the features of thepresent invention.

The advantages of the microprocessor of the present invention having asingle or unique program for each reaction compartment (and/or slidesupport element and/or reagent dispenser) eliminates the need for amicroprocessor which is able to plan the interdependent steps for aplurality of slides being processed, as required by prior art systems. Afurther advantage of having a separate micro-processing program uniqueto each reaction compartment (and/or slide support element, etc.), isthat if the programs of one or several reaction compartments fail, therewill be no effect on the operation of the other reaction compartments(or slide support elements). One advantage to the individualizedmicro-processing system contemplated above is that there is noappreciable downtime in the event of a failure in one or a few reactioncompartments (or slide support elements). To the contrary, in theinstruments of the prior art, if the microprocessor or operating systemfails, then the instrument is completely inoperable and must berepaired.

In the present invention, in a preferred embodiment, there can be acommon “master” operating system that could be in communication witheach individually unique program so that the user can open a separateprogram specific to any or all of the reaction compartments (and/orslide support elements) at any time. The separate individual programrunning a specific reaction compartment (and/or slide support elements)would have all the protocols loaded therein for completely processing amicroscope slide. The separate program could be updated and edited bythe user and with the help of the master program could update all theother separate programs so that each reaction compartment (and/or slidesupport elements) could have the same protocols updates or edits. In theevent of a master program failure, the separate unique programs to eachreaction compartment (and/or slide support elements) would still beoperational to process microscope slides; it just would lose the abilityof communicate with the separate programs of the other reactioncompartments (and/or slide support elements) for updating, downloading,or uploading information. In a variation of this, each reactioncompartment (and/or slide support elements) may be individuallyseparated and unique to itself with regard to its operating program withno link to the other reaction compartments (and/or slide supportelements). A further advantage to having a master operating system isthe ability to communicate with the other separate reaction compartment(and/or slide support elements) programs for diagnostic purposes,uploading, downloading, and general and specific communications betweenreaction compartments (and/or slide support elements).

In one embodiment of the present invention, all the motion controlrequirement necessary for operation of the system can be in the form ofAC, DC, solar, and optionally other power sources like pneumatic andsteam. The microprocessor can be run on AC, DC, and solar for example.The entire instrument is compact and can be configured with any amountor numbers of reaction compartments necessary. The instrument can beportable to be used in the field (research for example) or carried to anarea of use. The number of reaction compartments (and/or slide supportelements) typically would be 10-20 per chamber and are stackable or arejoined linearly or are connected in any other manner which isappropriate (e.g., see FIG. 3B). A portable field unit could have as fewas 1-5, or 5-10, reaction compartments (and/or slide support elementsper chamber), for example, for less weight. Preferably the componentsare made from light weight, anti-corrosive materials. A furtheradvantage of the present invention is that the instrument can beserviced in a modular approach. Each reaction compartment and/or slidesupport element and/or reagent pack support device in the module can beremoved individually and serviced or discarded and replaced with an allnew component. All the motion controls are preferably modular and eitherserviceable or completely replaceable. An advantage to this modularserviceability is that the other reaction compartments and/or slidesupport elements that are in use or could be used, are not affectedduring servicing of any device or part from a different reactioncompartment and/or slide support element.

An advantage of the present invention, as explained previously, is thateach microscope slide can be treated with a separate unique reagent,inferring that any microscope slide can have any reagent and be treatedat pressures and for varying amounts of treatment times which are thesame or different from any other microscope slide loaded into theapparatus. Examples of reagents which may be used in the presentinvention include, but are not limited to: antigen retrieval reagents,RNA and DNA probes, citrate buffer, EDTA, TRIS, PBS, with or withoutsurfactants or detergents like SDS, Tween, Brij, ionic and non-ionicdetergents, and silicone additives, rinse buffers, immunohistochemicalreagents, histochemical reagents, in-situ hybridization reagents, PCRreagents, coverslipping reagents, silicone oils, mineral oils, detectionreagents and processing reagents, liquid reagents, reconstituted dryreagents, biological reagents and aqueous and non-aqueous reagents, anddeparaffinizing compositions of water with one or more siliconesurfactants or silicone additives. Another advantage with the presentinvention is that cross contamination from reagents or biologicalspecimens on adjacent or nearby microscope slides is eliminated becauseeach microscope slide is separated and treated with its own reagent in aseparate reaction compartment or on a separate slide support element.

Another important advantage of present invention is that each individualreaction compartment and/or slide support element can be cleaned orrepaired separately and automatically at the same time that otherreaction compartments and/or slide support elements are being used toprocess microscope slides. Thus, there is no downtime or interruptionfor the other reaction compartments and/or slide support elements when aparticular individual reaction compartment and/or slide support elementis being cleaned or repaired. Each reaction compartment and/or slidesupport element can be separately cleaned and/or sterilized by steam,with or without a detergent or sterilizing reagent and dried with heated(below 100° C.) or superheated (above 100° C.) air. This type ofsterilized cleaning could be used for example if a biological specimenthat was being processed had infectious properties. Each reactioncompartment essentially has the properties of an individualself-regulated and controlled miniature autoclave. Sterilization of eachreaction compartment prior to use with the next biological specimenprocess can provide an inherent technical advantage due to theelimination of cross contamination and direct contact with infectiousbiological specimens. Sterilization can be performed using steam alone,or chemicals dispensed by a reagent pack or another dispensing element.

Pressure Cooker Method vs. Present Method

The regulation of pressure in the reaction compartments or pressurizablecommon chambers of the staining apparatus of the present invention isdifferent from that of a pressure cooker. A pressure cooker utilizeswater at the bottom of the cooker to produce steam to heat the insidechamber and produce the pressure inside the chamber. A pressure cookeris constantly producing steam and therefore is pressurized from initialheating thru cooling. This pressure is constantly being released througha vapor pressure release device. The mode of release can be a “rockervalve” that is set for a certain psig release by the “rocker valve”having a specific weight. When the pressure cooker's closed chamberbuilds up pressure that exceeds the weight of the “rocker valve”, thevalve unseats or opens the closed chamber until the pressure decreasesto a psig under the weight of the “rocker valve” This hissing that isnormally heard around a pressurized pressure cooker is very apparent.The hissing is the unseating of the “rocker valve” to release pressureexceeding the “rocker valve” weight. This is the regulator system of apressure cooker. Other models of commercial pressure cookers can alsouse a vapor pressure release device that are a pre-set one way valvethat releases pressure in the pressure cooker's chamber when thepressure exceeds the pre-set valve psig. In all pressure cookerscommercially known today there is always some way of releasing thepressure to maintain the pre-set limit of the pressure cooker. Thepressure limits of commercial pressure cookers are not adjustable; infact they are required to release the pressure in the chamber in acontrolled manner to keep the pressure at a constant psig which isusually 24-26 psig.

The present invention uses a separate source of pressure to pressurizethe individual reaction compartment or pressurizable common chamberbefore, during, or after the heating of the chamber or reagent. In thepresent invention, in this embodiment, maintaining pressure is in strictcontrast to commercial pressure cooker's maintenance of pressure. Thepresent invention doesn't release pressure to maintain a desired psig ortemperature requirement. The present invention can pressurize in a rangeof 0.001 psig to 5000 psig. The present invention maintains the pressureat a desired psig by not releasing any of the pressure in the chamber.The pressure of the present invention is maintained by modulating(turning off and on) the heating plate temperature and the amount ofpressure initially added to the individual reaction compartment or tothe pressurizable common chamber, an example being the pressurizablechamber of the present invention, whether it's an individual reactioncompartment holding a single microscope slide or biological specimencontaining vessel, or a pressurizable common chamber holding a pluralityof microscope slides or biological specimen containing vessels which ispressurized by a remote source of pressure initially and can beincreased (if desired) by the minimal evaporation of the reagentassociated with the biological specimen or microscope slide. Thepressure of the present invention is maintained by containing, notreleasing, the pressure generated during pressurization or heating. Thepressure source can be from the head space, as describe elsewhereherein, or from a remote source. If the present invention utilized themethod of a commercial pressure cooker to maintain and regulate pressureby producing pressure by evaporation and the subsequent release tomaintain a desired psig, the reagent(s) present individual reactioncompartments or pressurizable common chamber would go dry due to thecomplete evaporation of the reagent to produce the pressure thatultimately must be released to maintain the desired psig. The volume ofthe present invention typically utilizes very small amounts of reagentson the microscope slide to treat each slide (e.g., 0.1 microliters to5000 microliters, preferably 1 microliter to 3000 microliters). If thepressure produced by the reagent(s) evaporating was allowed to berelease from the present invention to maintain the desired psig, thesevery small amounts of reagent(s) present on the microscope slide wouldcompletely evaporate because the pressure they are producing is beingreleased to maintain the desired psig by using the commercial pressurecooker's maintenance method.

In a preferred embodiment of the invention, particular reagents aresupplied to the reaction compartment and/or slide on the slide supportelement from a reagent pack (also referred to herein as a reagentdispensing strip or pack) individualized for a single reactioncompartment and/or slide on the slide support element as described inmore detail in FIGS. 1-22 and 39-78 of Published PCT application WO2006/127852 and elsewhere herein (e.g., FIG. 40-42). Due to theextensive discussion of such reagent packs described therein, it is notconsidered necessary to provide further explanation in the presentdisclosure except to the extent that further embodiments or details ofoperation are newly presented herein.

While the invention is now described herein in connection with certainembodiments and examples so that aspects thereof may be more fullyunderstood and appreciated, it is not intended to limit the invention tothese particular embodiments and examples. To the contrary, it isintended to cover all alternatives, modifications and equivalents as maybe included within the scope of the invention as defined by the claimsbelow. Thus, these examples and embodiments, which include preferredembodiments, will serve to illustrate the practice of this invention, itbeing understood that the particulars shown are by way of example andfor purposes of illustrative discussion of various embodiments of thepresent invention for providing various principles and aspects of thepresent invention.

Moreover while various systems, devices, components, and apparatuses ofthe invention are described herein in particular embodiments andexamples, it is intended that all such systems, devices, components andapparatuses be interchangeable in regard to the various combinationsthereof which may be envisioned as embodiments of the inventiondescribed and claimed herein as long as such other embodiments which arenot explicitly herein function in accordance with the present invention.For example, the various types of reaction compartments, slide supportelements, heating elements, reagent pack support devices, dispensers,plungers, closure and sealing means, chambers, pressurizationapparatuses, and spreading devices, to list but a few, can replace eachother in various alternative embodiments of the invention.

Embodiments of FIGS. 1-6

Turning now to the figures, shown in FIG. 1 is a microscope slidestaining system designated by the general reference numeral 2. Themicroscope slide staining system 2 in a preferred embodiment comprises astaining apparatus 10, a remote reagent source 4 operatively connectedto the staining apparatus 10, a waste collection system 6 operativelyconnected to the staining apparatus 10, and a microprocessor operativelyconnected to the staining apparatus 10, and preferably to the remotereagent source 4 and waste collection system 6. The remote reagentsource 4 of the microscope slide staining system 2 preferably has aself-contained D.I. water, buffer, and/or reagent liquid production andmanagement module which is operatively attached to the stainingapparatus 10. The remote reagent source 4 is also referred to elsewhereherein as a “reagent module” or as a “remote reagent source”. Thisreagent module 4 can be plumbed to the staining apparatus 10 for“on-demand” efficient production of rinse buffers, antigen retrievalsolution, or any type of liquid reagent used in treatment of microscopeslides. The reagent module 4 can provide buffers or reagents like washrinses, antigen retrieval solutions, fixation solutions hydrationsolutions, dehydration solutions, mineral oil solutions, surfactantssolutions, ionic and or non-ionic additives solutions, buffer solutions,D.I. water rinses solutions, polyol additives solutions, alcoholsolutions, xylene solutions, limonene solutions, Tween solutions, Brijsolutions, and other reagents or solutions. The reagent module 4 canprovide liquids for use in the staining apparatus 10 by filling a bulkbottle, bottles, or storage reservoir to be used by the stainingapparatus 10. The bulk bottles would be operatively connected to eachset of reaction components or to each reagent dispenser or to adispenser of the X-Y-Z positioning device for use therein. The reagentmodule 4 can be connected to a known D.I. water source in the lab or canbe plumbed to a tap water source to produce D.I. water in-situ. Theregent module 4 may comprise reagent canisters (not shown) which areoperatively connected in a series or parallel for different types ofliquids to be dispensed in the staining apparatus 10. Different types ofreagent canisters can be employed by the reagent module 4 to producedifferent types of liquids for the staining apparatus 10. Each reagentcanister can produce its own “type” of “liquid” for use. The reagentmodule 4 preferably has a plumbed water supply, an electricalconnection, and conduits or plumbing to the staining apparatus 10 for aclosed system of operation. The reagent canisters may contain chemicalsin a solid, liquid, gel, semi-solid, colloidal, or any known physicalstate for treating a water source to produce a “ready to use” or “ondemand” production of reagents for the staining apparatus 10. Thereagent canisters of the reagent module 4 can be plumbed in a series orparallel to facilitate removal and replacement of the reagent canisterswhen the staining apparatus 10 is in operation. There can be two or moreof one specific “type” of reagent canister plumbed in a series orparallel on the reagent module to facilitate the removal of one emptycanister, while the other or others are still in operation. Preferably,operation of the staining apparatus 10 does not have to be stopped toadd or replace a reagent canister while the reagent module 4 is in use.The microprocessor 8 of the staining system 2 or a microprocessor in thereagent module can alert the technician to replace or remove a used orempty reagent canister. In a preferred embodiment of the reagent module4, the reagent module 4 is plumbed in line with a tap water source or DIwater source to the staining apparatus 10. The staining apparatus 10could use a salt-free rinse solution, for example, produced by thereagent module 4 comprising deionized water (DI water) with an ionicdetergent, non-ionic detergent, cationic detergent or surfactantpresent. The tap water plumbed to the reagent module 4 can be deionized,distilled, purified, and or sterilized by the reagent module 4 by UVirradiation, and/ or chemicals present in one of the canisters in thereagent module 4. If DI water is initially plumbed to the reagent module4, the DI water can be treated similar to non-DI water or tap water toproduce a very high quality sterile DI non-salt rinse with a surfactantpresent. The reagent module 4 may also be constructed to provide antigenretrieval solutions with different types of salts or surfactants knownin the art of antigen retrieval solution or antigen unmasking solutions.These chemical or solutions are well known in the art. Antigen unmaskingsolutions can be, for example, citrate buffer, EDTA, antigen retrievalsolutions having a pH in the range of 1-14, urea, with or withoutsurfactants or detergents like Tween, Brij, IGEPAL, SDS, glycols,polyols, alcohols, and other ionic or non-ionic surfactants ordetergents known in the art or others described elsewhere herein. Thisis a very convenient and economical way of providing these buffers orreagents “on demand” and delivering the buffers or reagents to theindividual reaction components of the staining apparatus 10 withoutstopping or interrupting the slide being processed on the stainingapparatus 10. The microprocessor 8 can alert the technician that one ormore reagent canisters in the reagent module 4 are to be removed orreplaced. The fittings on the reagent module 4 and reagent canistertherein can be of any type of “quick connect” or “quick disconnect”component know in the art for liquid distribution connections. Thisconcept of removing or replacing the reagent canisters “on the fly”without stopping the slide staining processes, complements the“independent access” of the staining apparatus 10 of the presentinvention. All prior art automated slide strainers have to, at sometime, stop the slide staining process to either replenish, replace, oradd reagents to their staining apparatus 10 during slide processing orbefore starting a new staining protocol . This embodiment of the presentinvention eliminates the need to stop the staining apparatus 10 merelyto replace, change, or refill reagents required to stain a biologicalspecimen on a microscope slide while the staining apparatus 10 is inoperation processing at least one biological specimen on a microscopeslide.

In a further embodiment of the present invention, as indicated in FIG.1, the microscope slide staining system 2 comprises a self-containedwaste collection system 6 also referred to herein as a “waste module 6”or “waste management module”. This waste module 6 is operativelyconnected to the staining apparatus 10 for treatment of hazardous wastesor biological wastes or other wastes produced therein. The waste module6 treats “on demand” both solid and liquid wastes. The waste module 6preferably can separate liquid waste from solid wastes. The waste module6 can treat the solid and liquid waste to produce non-hazardous wastethat can be disposed by the laboratory disposal services. The wastemodule 6 preferably can separate hazardous waste from non-hazardouswaste. If a hazardous waste can't be decontaminated by the waste module6, the module will place the solid or liquid non-treatable waste into asealed container (not shown) that can be disposed by lab personnelwithout the need to place the removable and disposable hazardous wastecontainer in any other container for disposal. The waste container to bedisposed will have fitted on its self a “break-away” fitting to seal thewaste container from the lab's environment. The waste module 6 ispreferably plumbed in a series or parallel to provide waste managementwhile the staining apparatus 10 is in operation. The waste module 6 candecontaminate several hazardous wastes like, but not limited to DAB,Fast Red, Special Stains, Xylene, alcohol, chromogens, reagents,buffers, infectious and biological waste, etc. Each hazardous chemical,liquid, gas, or solid can be decontaminated by its own decontaminatingcanister or non-treatable waste can be separated into disposable wastecanisters. Each decontamination canister can be separately removed orreplaced on demand without stopping the staining apparatus 10 duringoperation.

The schematic in FIG. 1 is intended to representative of any microscopeslide staining system contemplated herein and may comprise components ofany of the invention embodiments described or contemplated herein in anycombination which functions in accordance with the treatment, staining,and pressurization aspects of the present invention.

Shown in greater detail in FIG. 2 is the staining apparatus 10 of themicroscope slide staining system 2 of the present invention. Stainingapparatus 10, shown in FIG. 2, is substantially the same as the singlechamber 282 shown in FIG. 85 of the parent application U.S. Ser. No.11/439,722, comprises a top upper wall 12, a bottom wall 14, first sidewall 16 and second side wall 18. The staining apparatus 10 furthercomprises a front wall 20 (FIG. 3A), a back wall 22 (FIG. 4), and aninner space 24. Contained within the inner space 24 is a plurality ofsets of reaction components 26 (also referred to herein as reactionmodules). Figs.2-6 and 34-36 show six sets of reaction components 26a-26 f in the staining apparatus 10, but this is for illustration only.In other embodiments of the staining apparatus 10 any number of sets ofreaction components 26 may be present, for example, less than or morethan 6, such as 4 to 50 sets of reaction components 26.

Each set of reaction components 26 a-26 f comprises, in the embodimentof the invention of FIG. 2, a reaction compartment 30, a slide supportelement 32 for supporting a microscope slide 48, a reagent pack supportdevice 34 for supporting a reagent pack 42, and a dispenser plunger 36for causing expulsion of a reagent from a reagent container 40 of thereagent pack 42 onto the microscope slide 48. The dispenser plunger 36(also referred to herein in some embodiments as a dispensing element)may move in an upward or downward direction for being positioned todispense a reagent 38 onto the microscope slide 48. For example, in FIG.2, reaction components 26 a and 26 c show the dispenser plunger 36forcing the reagent 38 from the reagent container 40 of the reagent pack42 which is positioned on a reagent pack support device 34. The reagent38 is forced through a reagent conduit 44 of the reaction compartment 30into an inner space 46 thereof onto a microscope slide 48 placed uponthe slide support element 32. The dispenser plunger 36 is then withdrawnfrom the reagent container 40 (as shown for reagent components 26 b and26 e). In some cases the dispenser plunger 36 is able to cause expulsionof a reagent 38 from the reagent pack 42, and is also able to separatelydispense a reagent delivered from a remote reagent source 4. In otherembodiments, these functions may be performed by separate devices suchas the dispensers 319 and 320 described in further detail below inregard to FIG. 29A for example. The microscope slide 48 may be heated bya heating element 50 which is positioned on the slide support element 32underneath the microscope slide 48. As is indicated in FIG. 2, each setof reaction components 26 can be in a different phase of operationindependently of each other. For example, in FIG. 2, reagent 38 is beingdispensed onto the microscope slide 48 in reaction components 26 a and26 c. Reagent 38 is being removed from slide support element 32 bytilting thereof in reaction components 26 b. In reaction components 26d, the inner space 46 of the reaction compartment 30 has been floodedwith a reagent 38 for treating or rinsing the microscope slide 48. Inreaction components 26e, the dispenser plunger 36 has been removed fromthe reagent pack 42, reagent 38 has been removed from the microscopeslide 48 and the slide support element 32 is in an “upright” positionfor allowing further treatment or disposition of the microscope slide48. In reaction components 26 f, the reagent pack 42 has been removedfrom the reagent pack support device 34 and the slide support element 32is without a microscope slide 48.

FIG. 3A shows a perspective view of the embodiment of the stainingapparatus 10 of FIG. 2. The front wall 20 of the staining apparatus 10comprises a plurality of slide support element doors 54 which can open(see FIG. 5) to allow the slide support elements 32 to be ejected fromthe staining apparatus inner space 24, or returned to the inner space24. Similarly, the front wall 20 of the staining apparatus 10 comprisesa plurality of reagent pack support device doors 56 which can open toallow the reagent packs 42 to be inserted into or ejected from thereagent pack support devices 34 for use in a treatment protocol, orafter such use. Additionally, the reagent pack support device 34 can beconstructed so as to be able to be ejected from the staining apparatus10 through the door 56 (or without a door) for removal of a reagent pack42 therefrom, or for placement of a reagent pack 42 thereon. The reagentpack support device 34 can then be returned (reinserted) into the innerspace 24 of the staining apparatus 10 for treatment of the microscopeslide 48 on the slide support element 32. The reagent pack supportdevice 34 can, in an alternate embodiment, be positioned inside thestaining apparatus 10 and only the reagent pack 42 is inserted into thereagent pack loading/removal opening (with or without an access door 56)wherein the reagent pack support device 34 “captures” or “grabs” thereagent pack 42 and pulls the reagent pack 42 into the stainingapparatus 10 (like a CD-player in an automobile). When the reagent pack42 is inside the staining apparatus 10 the microprocessor can recognizethe reagent pack 42 and initiate the particular treatment protocolassociated with that reagent pack 42. The reagent pack 42 may moveoutside and inside the staining apparatus 10 during staining to line upthe desired reagent container thereon or can remain entirely inside thestaining apparatus 10 during staining and only be moved outside of thestaining apparatus 10 when ejected for disposal.

The staining apparatus 10 of FIG. 3A further comprises one or more ofindicator lights, buttons, or gauges 58 and at least one display panel59 which correspond to a particular slide support element, reactioncompartment or reagent pack or reagent pack support device. For example,one indicator light, button (e.g., an eject/insert button), or gauge 58may be used to cause a door 54 or 56 to open or close, or slide supportelement 32 to be inserted, or may indicate that the component is “on” or“off”, or may indicate some physical parameter associated with thecomponent, such as its temperature, pressure, or operational status. Thedisplay panel 59 may show the status or identify of the treatmentprotocol or reagent 38 to be used or currently in use in the reactioncompartment 30.

Shown in FIG. 3B is a multi-staining apparatus version of the microscopeslide staining system of the present invention designated by referencenumeral 52 which comprises four staining apparatuses 10. Each stainingapparatus 10 is indicated as containing 6 sets of reaction components26. As noted above, the staining apparatus 10 may contain any number ofsets of reaction components 26 (also referred to herein as reactionmodules), for example from 4-50 sets and each microscope slide stainingsystem of the invention may comprise one or more staining apparatuses10. For example, in the microscope slide staining system 2 of thepresent invention, a single staining apparatus 10 may comprise theentire treatment unit of the staining system of the invention. Thestaining apparatus 10 may be arranged vertically of horizontally or inany configuration suitable for operating and the staining apparatus 10may be constructed so that the sets of reaction components 26 arearranged in an arcuate pattern relative to one another within thestaining apparatus 10 rather than linear.

Shown in FIG. 4 is a top plan view of the staining apparatus 10 of FIGS.2-3B comprising reaction components 26 including reaction compartments30, and slide support elements 32. Each slide support element 32 hassealing means 60 (such as O-rings or ground glass surfaces). Each slidesupport element 32 in this embodiment is connected by a shaft 62 to amotor 64 for pushing the slide support element 32 in a forward directionfor ejection from the inner space 24 of the staining apparatus 10 and/orreaction compartment 30, for loading or removal of a microscope slide48, or in a reverse direction for retracting the slide support element32 into the staining apparatus inner space 24 and/or the reactioncompartment 30 for treatment of the microscope slide 48. Optionally,each slide support element 32 may have a handle 66 for manually pullingor pushing the slide support element 32 into or out of the stainingapparatus 10.

FIG. 5 shows the first, second, and fourth slide support elements 32from the left ejected from the staining apparatus 10, in positions forplacement of microscope slides 48 thereon. FIG. 6 is a top plan view ofthe staining apparatus 10 of FIG. 5 wherein the first, second and fourthslide support elements 32 from the left are shown in a placement orremoval position outside of the inner space 24 of the staining apparatus10. The motors 64 have caused extension of the shafts 62 causingexpulsion of the slide support elements 32 from the correspondingreaction compartments 30, which in this embodiment are preferablyopen-ended.

The slide support elements of the invention preferably can beautomatically moved in any position while the reaction compartment ispressurized (positively or negatively) with or without a heating meansto heat the reagent under said positive or negative pressure. Saidheating means can be a conductive, convective, and/or radiant heatingelement incorporated in or adjacent to the slide support element forheating the microscope slide and a biological specimen thereon. Theslide support elements can be moved independently forward, backward,rotated along a longitudinal axis, and/or tilted, while the reactioncompartment's inner space in under positive or negative pressure. Suchmovement of the slide support element, does not cause the positive ornegative pressure to be expelled or otherwise “leak out” of the innerspace of the reaction compartment since the slide support element issealed therein. The seal of the slide support element to the reactioncompartment causes retention of the pressure (positive or negative) heldin the inner space of the reaction compartment during movement of theslide support element in within the reaction compartment. This movement,which does not alter the pressure in the reaction compartment, would beadvantageous when it is desired for the microscope slide to be movedwhen the reaction compartment is under positive or negative pressure,e.g., during a treatment protocol. For example, the reagent contactingthe biological specimen on the microscope slide present on the slidesupport element can be mixed or agitated by mechanical movement of theslide support element under positive or negative pressure. This movementfor mixing the reagent on the microscope slide can be, for example, aforward and alternating backward movement along with a tilting from sideto side movement to cause a circular rotation of the reagent on themicroscope slide. Further, the microscope slide can be rotatedcompletely or partially to an upside down position (0° to 180° from itsoriginal upright horizontal staining position, for example) and rinsedunder pressure to remove the reagent on the microscope slide. Anyprotocol step requiring movement of the slide support element underpositive or negative pressure is contemplated. The movement contemplatedabove can be employed for mixing, rinsing, or otherwise treating themicroscope slide with a protocol that has at least one step whichbenefits from, requires, or otherwise needs the microscope to be movedor mobile under positive or negative pressure, for example, for rinsingthe slide and retaining the slide to the main treatment position. Italso preferred that the slide support element is moveable at any timeunder or not under pressure. The slide support element can be movedforward, backward, and rotated 360° in relation to the stationaryreaction compartment. Alternatively, the reaction compartment can alsomove relative to the stationary slide support element in a forward,backward, or 360° rotational movement.

In the staining apparatus of any of the embodiments contemplated hereinthe chamber may be constructed so that a portion of the front wall,upper wall, bottom wall, back wall, and/or side walls, can be detachedor opened to enable access to the inner space of the staining apparatusfor removal, replacement, repair, or insertion of any of the reactioncomponents therein. For example, a portion of the front wall in front ofa slide support element or reaction compartment can be removed to enablereplacement thereof, without having to access or disturb other sets ofreaction components.

In an alternate embodiment of the invention, the apparatus is anautomated biological processing instrument having a pressurizable commonchamber (e.g., see FIGS. 35 and 36) that can hold a plurality ofmicroscope slides on a plurality of independently movable slide supportelements in the pressurizable common chamber, wherein the slide supportelements are automatically and independently movable inside theprocessing chamber while the common chamber is under positive ornegative pressure with or without heating means to heat reagents on themicroscope slides while under said positive or negative pressure. Saidheating means can be a conductive, convective, and/or radiant heatingelement incorporated in or adjacent to the slide support element. Themovements of the slide support elements are independent of each otherwhile in the pressurizable common chamber and microscope slides thereinare under positive or negative pressure. In an alternate embodiment, theslide support elements are positioned on a common platform which ismovable, wherein a plurality of slide support elements under positive ornegative pressure are movable together with or without heating means toheat reagents disposed on the microscope slides.

The automated biological processing apparatus contemplated herein canhave movable biological processing devices (e.g., reagent dispensers)that move over or around the microscope slides on the slide supportelements whether the microscope slides and slide support elements aremovable (or in movement) relative to the processing devices or are in afixed position while being under positive or negative pressure (and withor without a heating means to heat a reagent associated with themicroscope slide). As contemplated herein, said biological processingdevices can be (but are not limited to) reagent-air mixing gas jets,rinse dispensers, air knives for blowing off reagents, reagentdispensers to dispense reagents (such as antibodies, stains, molecularprobes, detection reagents, RNA probes, DNA probes, in-situhybridization reagents, evaporation inhibition oils, or detectionreagents or other reagent elements contemplated herein), OpticalRecognition Characters (ORC) code readers, machine readable devices toread codes or symbols, reagent spreaders, or any other processingdevices known in the art of processing biological specimens onbiological supports.

In preferred embodiments, the present invention comprises automatically,independently, and/or simultaneously movable slide support elementsand/or automatically, independently, and/or simultaneously movablebiological processing devices and/or reaction compartments, underpositive or negative pressure which are operable while a reagentassociated with a biological specimen on the microscope slide on theslide support element is being heated by a conductive, convective,and/or radiant heating element incorporated into or adjacent to themoveable slide support element and/or movable biological processingdevices and/or movable reaction compartments. All such movablecomponents present inside the staining apparatus of the apparatus canautomatically move under positive or negative pressure wherein a reagentassociated with a biological specimen on the microscope slide on theslide support element is heated by heating means present in or adjacentto the slide support element or microscope slide. Preferably thesemovements of the movable components present in the staining apparatus(or reaction compartments) under positive or negative pressure, with orwithout heat, do not release or otherwise change the positive ornegative pressure within the staining apparatus (or reactioncompartments) while the components are in motion.

In an alternate embodiment of the invention, represented for example inFIGS. 34 and 35, the staining apparatus of the invention may comprise anX-Y-Z positioning device. X-Y-Z positioning devices are commonly used inthe art of dispensing reagents to microscopes slides and otherbiological substrates. One commercially available X-Y-Z positioningdevice can be obtained from Tecan Group Ltd., 103 CH-8708 Mannedorf,Switzerland. The X-Y-Z positioning device comprises a movable head as adispensing component and can be used to dispense reagents to themicroscope slide on the slide support element outside or inside of thereaction compartment as described elsewhere herein. The X-Y-Zpositioning device is able to move the movable dispensing componenthorizontally, laterally, or vertically to enable the movable dispensinghead to be used as a dispensing device independently of the reagent packor as an adjunct thereto.

This embodiment may use the reagent pack to dispense reagents inaddition to reagents dispensed directly from the X-Y-Z positioningdevice. The dispensing head of the X-Y-Z positioning device can dispensereagents through conduits therein, as for the dispensing plunger whichcauses expulsion of reagents from the reagent pack. The dispensing headmay comprise a distal portion forming a dispensing head which may have apipette attached for dispensing reagents from an array of containers orthe distal position could have disposable pipette tip attached that canbe used and removed between application of each reagent. The dispensinghead of the X-Y-Z positioning device can, in one embodiment, be used asthe dispensing plunger to dispense reagents from the reagent pack or asan inkjet printer, an optical code reader, a scanner, or an aspirator.

All the heating elements of the present invention (e.g., the slideheater, reaction compartment heater, dispensing port heater, cavityheater, reagent strip holder heater, and the slide support heater(described below)) can be adapted to heat and sustain heating from about1° C. to about 1000° C. The temperature of the reagent in the reactioncompartment can be in the range of ambient, (25° C.) or heated to 100°C. or greater. The reagent is preferably in the range of 25° C. to 400°C., and is more preferably in the range of 25° C. to 150° C. Thetemperature of the reagent when heated is preferably in the range of100° C. to 160° C. More preferably, the temperature of the reagent is inthe range of 101° C. to 150° C. More preferably, the reagent temperaturewould b in the range of 110° C. to 130° C. The reaction compartment canbe pre-pressured by a separate gas source described in US PatApplications 20060281116, 20060275889, and 20060275861 or the pressuredgas can be produced by the compression of the “head space” of thereaction compartment described in detail below. The pre-pressure gassent to the reaction compartment is also known as “pre-reactionpressure” The reaction compartment can have further pressure producedfrom the evaporation of the liquid reagent present on or around themicroscope slide. An example being, the separate source of gas beingbrought to the reaction compartment to pre-pressurize the reactioncompartment is say 25 psig. The heating source would heat the reagentaround the microscope slide and/or heat the reagent on the microscopeslide. The evaporating reagent around the microscope slide and/or thereagent on the microscope slide produced an additional 5 psig, forexample. The total psig, for example, would be the initial 25 psig fromthe pre-pressure source plus the psig from the evaporated reagent totals30 psig. This addition of the separate source of gas and the addition ofthe evaporated reagent total is known as the “total reaction pressure”or “TRP”. The evaporating reagent producing pressure is directly relatedto the type of reagent being heated and its evaporation characteristics.The reagent evaporation pressure can be in the range of 0-10 psig, forexample. Pressures for pre-pressurization, regular pressurization orin-situ pressurizations can be in the range of 0.01 PSIG to 1000 PSIG,more preferably in the range of 1 to 500 PSIG, and still morepreferably, in the range of 10 to 150 PSIG.

In an alternate embodiment of the present invention, each of the slidesupport elements can support at least two microscope slides. Preferablyeach separate microscope slide would be heated by separate heatingelements, but they could be heated by a common heating element. Thisslide support element which is able to hold at least two microscopeslides would have a single reaction compartment for pressurizedtreatment of the at least two microscope slides on the single slidesupport element. This embodiment is optimally used in high volumemicroscope slide testing laboratories, wherein instead of one slide perslide support element and each slide support element having its ownsingle reaction compartment, this embodiment would result in a decreaseof the number of slide support elements which carry only a singlemicroscope slide per slide support element. For example, in thisembodiment, the staining apparatus could comprise 10 slide supportelements each able to hold at least two slides. If the stainingapparatus had 10 of these double slide support elements these 10 slidesupport elements would support 20 slides together if they were all inuse for an initial treatment run of these 20 microscope slides.Preferably the staining apparatus would also comprise a plurality ofslide support elements which support single microscope slides enablingthe addition or removal of single slides onto and into the stainingapparatus for independent access to the staining apparatus. Thus,individual single slides could be inserted or removed from the stainingapparatus while the other double slide supports were already inoperation. For example, in one embodiment of a staining apparatus ableto treat 40 slides, there could be 10 double slide support elementsholding 20 slides, and 20 single slide support elements for independentaccess to 20 slides enabling a total of 40 slides to be treated. Thestaining apparatus could comprise any combination of double, triple, (ormore) slide support elements along with a plurality of single slidesupport elements in any combination of single or plural slide supportelements. As stated above, if a slide support element is sized tosupport 2 or more microscope slides, this slide support element wouldhave its own reaction compartment unique to itself for treatment of theslides thereon. If a movable slide support element, for example, held 3microscope slides, this slide support element would be associated withits own reaction compartment for the pressurized treatment or treatmentof the 3 slides present thereon. In an alternate embodiment, thestaining apparatus may comprise separate reaction compartments thatseparately enclose or at least partially enclose the at least two ormore microscope slides on the single slide support element, therebyenabling separate treatment of the at least two or more microscopeslides even though they are movable together on the common slide supportelement. In an alternative embodiment the at least 2 microscope slidesupport elements can be moved into or out of a staining apparatus havinga pressurizable common chamber for treatment of the microscope slidesalong with any single slide support element that is also capable ofmoving into or out of the staining apparatus with the pressurizablecommon chamber.

Embodiments of FIGS. 7-22B

Shown in FIGS. 7-22B are reaction components 104 of a staining apparatus100 such as staining apparatus 10 of an analytic apparatus of thepresent invention having a cylindrical reaction compartment 112, a slidesupport element 114, and a reagent pack support device 116 forsupporting a reagent pack, such as previously described elsewhereherein. Preferably, the reaction compartment 112 has an inner diameterof 1.5-5 cm, and more preferably 2-3 cm, and more preferably 2.5-2.8 cm,and has a wall thickness of 2 mm to 3 mm. The length of the slidesupport element 114 is preferably 10-20 cm, and more preferably 12-15cm. The length of the reaction compartment 112 is preferably 15-30 cm,and more preferably 18-22 cm. The reagent pack support device 116 inthis embodiment is operatingly connected (e.g., attached at a top) tothe reaction compartment 112 via a reagent conduit 122 in the reagentpack support device 116 or reaction compartment 112 which opens to aninner space 120 of the reaction compartment 112. There is an injectorport orifice 124 in the reagent pack support device 116 which is adaptedto receive an injector nozzle or port from a reagent container of areagent pack 106. The reagent pack support device 116 has a front end126 and a rear end 128. The reagent pack support device 116 functions toreceive, support, move and eject a reagent pack 106 of the presentinvention and preferably can move upwardly and downwardly and forwardand backward. The slide support element 114 has a base 134 which canreciprocatingly move into and out of the reaction compartment 112 andinto or out of the staining apparatus 100. The slide support element 114comprises a heating element 136 upon which a microscope slide 140 (likemicroscope slide 48) is placed. The slide support element 114 mayoptionally have a handle 142 which enables a technician to manuallyinsert and withdraw the slide support element 114 from the reactioncompartment 112 and staining apparatus 100. The slide support element114 preferably further comprises a sealing means which in the embodimentof FIG. 7 is a front O-ring 144 and a rear O-ring 145 for providing apressure resistant seal of the base 134 against the inner surface 118 ofthe reaction compartment 112. Other embodiments of sealing means whichcan be employed in the invention are described elsewhere herein. Theslide support element 114 (and base 134) can be constructed frommaterials which include, but are not limited to, glass, quartz, Pyrex®,borosilicate, steel, metals, aluminum, composites, polymers such aspolycarbonate and plastics or combinations thereof.

The slide support element 114 also preferably has a drainage port 146for receiving and draining reagents and waste liquids from the reactioncompartment 112. The slide support element 114 further preferably hasone or more cooling ducts 148 which are operatively connected to a subheating element cooling space 148 a beneath the heating element 136, andone or more cooling duct exits 148 b which evacuate the cooling air orliquid from the sub heating element cooling space 148 a. The slidesupport element 114 preferably further comprises a first air/pressureduct 150 and a second air/pressure duct 152 for regulation of thepressure within the reaction compartment 112 as discussed elsewhereherein. The duct 150 and/or duct 152 or an additional duct (not shown)can be used for releasing and/or regulating pressure from the reactioncompartment 112. The slide support element 114, as noted above,comprises a heating element 136 upon which the microscope slide 140 isplaced for application of reagents thereon. The reaction components 104may further comprise a thermocouple or other temperature measuringdevice for measuring temperatures of the slide or other componentstherein. Before operation the slide support element 114 is inserted by asliding motion into the inner space 120 of the reaction compartment 112(see FIG. 8A-8B). Also before operation the reagent pack 106 (or anyother reagent pack described or enabled herein) is inserted into thereagent pack support device 116, for example, inserting a first end 43of the reagent pack 106 into the front end of 126 of the reagent packsupport device 116, wherein during operation the reagent pack 106 ismoved in a direction toward the rear end 128 of the reagent pack supportdevice 116. The reagent pack 106 may be advanced manually orautomatically via a pulling or pushing device, including rollers or atrack which incrementally advances the reagent pack 106 as instructed bya microprocessor. The reaction compartment 112 further comprises areagent conduit 122 (like reagent conduit 44) for allowing passage of areagent from the reagent pack 42 into the reaction compartment 112. Thereaction components 104 also comprise a dispenser plunger 154 (alsoreferred to herein as a dispensing element and similar to dispenserplunger 36 above), which has a dispensing canal 156 therein for allowingpassage of another reagent or solution therethrough preferably from aremote source. The reagent pack support device 116 preferably has aninjector port orifice 124 for receiving at least a portion of aninjector nozzle 46 from a reagent container 107 of the reagent pack 106during use thereof. The staining apparatus 100 may comprise a separatedevice (other than a dispensing element) for pressing reagents from thereagent pack 106 such as shown in the embodiments of FIGS. 29A-33H.

During operation, as shown in FIGS. 8A-8B and 13, a reagent pack 106 (orany other reagent pack described or enabled elsewhere herein) isinserted through a door not shown in front wall 102 into the reagentpack support device 116 as previously described and a reagent container107 is positioned over the injector port orifice 124. The dispensingplunger 154 is extended downwardly into the reagent container 107 of thereagent pack 106 wherein it engages a piston, forcing the pistondownwardly and causing ejection of the reagent 38 from the container 107through the reagent conduit 122 and providing reagent 38 deposited ontothe microscope slide 140. When the dispensing plunger 154 forces thepiston 44 downwardly, a seal is maintained within the reagent container107 and in a preferred embodiment enables maintenance of pressure withinthe reaction compartment 112. The reagent 38 can be mixed on themicroscope slide 140, for example, by delivering bursts of air 162through the first air/pressure duct 150 and the second air/pressure duct152 as discussed in further detail below. In a subsequent step thedispensing plunger 154 may be withdrawn (FIG. 9A-9B) and the base 134 ofthe slide support element 114 tilted within the reaction compartment 112to allow the reagent 38 to drain from the microscope slide 140, forminga reagent drainage 160 which is collected in the drainage port 146,removed from the reaction compartment 112, and collected in a wastestorage container (not shown). In a later step (FIGS. 10A-10B) the slide140 is returned to an upright, horizontal position and the reagent pack106 is advanced until the rinse port aperture 108 in the reagent pack106 is positioned above the injector port orifice 124 wherein rinsesolution 163 is delivered from a rinse solution reservoir (not shown).Furthermore, air or liquid may be delivered through the dispensing canal156 in the dispensing plunger 154 to cause mixing of reagent 38 or toremove the reagent 38 from the microscope slide 140, or to enhance therinsing of the reagent 38 or rinse solution 163 from the microscopeslide 140 (e.g., see FIGS. 11A-11B). Finally as shown in FIG. 12, afterall reagents from the reagent pack 106 have been dispensed, the portionof the slide support element 114 which carries the microscope slide 140is withdrawn from the reaction compartment 112 wherein the microscopeslide 140 is then removed from the slide support element 114. Note thatFIGS. 13-14 are enlarged versions of FIGS. 8A and 10A, respectively andare provided herein for the purpose of more easily showing the stepstherein.

FIGS. 15A-16B provide a more detailed description of how the bursts ofair 162 delivered form the first air/pressure duct 150 and secondair/pressure duct 152 can be used to cause mixing of the reagent 38 onthe microscope slide 140. Preferably, the first air/pressure duct 150and second air/pressure duct 152 are operated alternately to providebursts of air 162 in alternating clockwise/counterclockwise directionsto agitate the reagent 38. The first air/pressure duct 150 and secondair/pressure duct 152 can also be used to pressurize the reactioncompartment 112. At any desired time the heating element 164 can be usedto heat the slide 140 and reagent 158 thereon as discussed in greaterdetail elsewhere herein. As shown in FIGS. 17-19B, after the microscopeslide 140 is heated, it can be rapidly cooled by directing air or liquidvia the cooling ducts 148 into sub heating element cooling spaces 148 awhich are located below the heating element 164 which in one embodimentis located below and is used to heat a hot plate 166 upon which theslide 140 is positioned. Air or liquid used for cooling can then passthrough cooling duct exits 148 b. In another embodiment, shown in FIGS.20-22B a sub heating element cooling space 148 c is similar to subheating element cooling space 148 a except the cooling air or liquidwhich passes through the sub heating element cooling space 148 c isdelivered via one of the cooling ducts 148 and exits the slide supportelement 114 via the outer cooling duct 148.

As shown in FIGS. 7-22B, each reaction compartment 112 of the stainingapparatus 100 preferably comprises a hollow cylinder, preferablyconstructed of a thermoplastic resin or polymer (including but notlimited to polycarbonate or any other polymeric material able towithstand elevated temperatures and pressures), glass, Pyrex®, quartz,other crystalline materials, and metals and metal alloys. The tubularnature of the reaction compartment 112 is preferred because the elevatedpressures created within the reaction compartment 112 during its use aremore evenly distributed therein.

The seal between the outer surface of the slide support element 114 andthe inner surface of the reaction compartment 112 can be formed usingO-rings, as shown in the FIGS. 23-38B or can be formed using aninflatable O-ring, a seal, or an inflatable seal depending on the shapeof the mating surfaces. The sealing means can be constructed of plastic,polymer, thermoplastic, resin, ceramic, rubber, metal glass, orcomposite, for example.

Or in a preferred embodiment, sealing surfaces comprising an outersurface portion of the slide support element 114 and an inner surfaceportion of the reaction compartment 112 are made of a low toleranceground or polished sealing surface. These sealing surfaces when engagedfrom a seal which replaces and eliminates the need for a ring seal orinflatable or seal raised above the mating surfaces. In this embodiment,the ground or polished mating surfaces alone, when joined together,produce a microscopic seal with a large surface area to seal themicroscope slide within the reaction compartment 112 and which is ableto maintain an elevated pressure therein (above atmospheric) even underhigh temperature conditions above 100° C. The material of the slidesupport element 114 and the tubular reaction compartment 112 can featurea very high tolerance ground or polished seal on the mating surfaces. Inthe preferred embodiment, the slide support element 114 and the reactioncompartment 112 are made of a high tempered glass material like Pyrex®,or any material that can produce a ground or polished mating surface toform a seal which maintains a pressure above atmosphere pressure. Theground glass surface, or polished surface of the slide support element114 against the ground or polished surface of the reaction compartment112 yields an air-tight and pressure-tight seal when the two ground orpolished surfaces are joined together, such that, there is no void spacewhich must be filled by a raised surface such as an O-ring. Thisembodiment of the present invention thus eliminates the need for raisedseals (e.g., O-rings) thus reducing maintenance cost for the replacementof separate seal components such as O-rings and increases simplicity andefficiency and seals the reaction compartment even under pressures aboveatmospheric levels (e.g., above 14.7 psig (101.325 kPa), i.e., above 0psig (101.325 kPa)) and high temperature conditions above 100° C.

As noted herein, the staining apparatus (e.g., staining apparatus 10 or100) of the staining apparatus of the present invention preferablycomprise a plurality of sets of reaction compartments 112, such as shownin FIG. 7. Each set of reaction components 110 comprises a tubularreaction compartment 112 (although the reaction compartments may not betubular, but may be rectangular, a slide support element 114 and in aparticularly preferred embodiment a reagent pack support device 116. Thereaction compartment 112 has an inner surface and an inner space intowhich the slide support element 114 can be moved for treating abiological sample on a microscope slide 140 thereon. The slide supportelement 114 is able to slide into and out of the reaction compartment112 in a manner similar to a piston within a cylinder. When the slidesupport element 114 is withdrawn from the reaction compartment 112and/or from the staining apparatus 100, a microscope slide 140 can beplaced thereon or removed therefrom. The slide support element 114 canbe inserted into the reaction compartment 112 for treatment of thebiological sample on the microscope slide 140 as described elsewhereherein. As shown below, the slide support element, in a preferredembodiment) can be turned (tipped or rotated) within the reactioncompartment 112 for facilitating the removal of reagents or fluids fromthe microscope slide 140 after the microscope slide 140 has beentreated, as shown in the figures (e.g., see FIG. 9B). Reagents or fluidson the microscope slide 140 can be mixed by air circulation as shown inFIGS. 15A-16B for example or by rotational movement of the slide supportelement 114. After heating, the microscope slide 140 can be cooled bycirculation of air or fluid thereunder, for example as shown in FIGS.18A-22B. In another embodiment, the microscope slide 140 could be cooledby using a circulating liquid such as a reagent that becomes pre-heatedby passing under the heated slide thus transferring heat to thecirculating reagent which could then be dispensed onto the microscopeslide 140.

The reaction compartment 112 of the present invention (or other reactioncompartments) can be constructed of any material known in the art ofhigh temperature and pressure compatible devices. These materials alsoinclude, but are not limited to, plastics, polymers, composites,ceramics, glass, quartz, metals and coated metals. The components of thereaction components 112 can be coated for resistance to porosity, toincrease hydrophobic and hydrophilic properties, for ease of cleaning,chemical resistance, and stain resistance. These coatings could be, forexample, Teflon®, fluoropolymers, any other known coating that wouldimpart these desirable properties to all surfaces of reactioncompartment 112 and slide support elements 114 and surroundingstructures with a different coating being present on different portionsof the apparatus. In one embodiment, for example, the inner surface ofthe reaction compartment 112 and outer surface of the slide supportelement 114 may be coated with a hydrophobic, chemical, and stainresistant coating to aid in the draining of the condensed reagents onthe inner surface of the reaction compartment 112 or outer surface ofthe slide support elements 114 and ease of removal of reagentstherefrom.

The slide support element 114 preferably has incorporated therein aheating element 136, and a hot plate (which may be one and the same) andwhich may include guide clips or pegs or elements to position and securethe microscope slide thereon. The tops of the clips may be positioned tobe below an upper surface of the microscope slide, so as to preventreagent on the slide 140 from being wicked off by the clips by capillaryaction.

In a particularly preferred embodiment, underneath the heating element136 is one or more recessions (sub-heating element cooling spaces 148 a)which are connected via cooling ducts 148 to a gas or liquid supplysource to quickly cool the heating element 136 thereby quickly coolingthe microscope slide and the reagent thereon.

The slide support element 114 and reaction compartment 112 can beconstructed of any material suitable for use under pressurizedconditions and resistant to corrosion by laboratory reagents, includingbut not limited to stainless steel, metals, plastics (clear or opaque),polymers (e.g., polycarbonate), tempered glass, and Pyrex® or othermaterials mentioned herein.

Containment of waste and used reagents from the staining apparatus willbe now briefly discussed, (see further discussion above).

In a preferred embodiment the staining apparatus of the presentinvention (e.g., as represented in FIG. 1) has a waste collection system6 which is operatively connected to the reaction components of thestaining apparatus 10 by one or more fittings that can join multipletubes or conduits. In a preferred embodiment of the present invention,this main fitting (not shown) can be joined to the waste container ofthe waste collection system (waste module) 6 (which may be disposable ornon-reusable) by a breakable joint present on the waste container. Thisfitting on the waste container snaps together with the main fitting ofthe instrument. This attachment is secure and will not leak underpressure. When detached, this fitting on the waste container partially“breaks away” and leaves behind on the waste container an airtight,leakproof, tamper proof, non-removable seal. The residual piece that wasdetached from the waste container is removed by the technician and thenis ready to be reattached to a new waste container. The waste containeris now ready to be disposed of in its entirety by a technician ormedical waste personnel. The tamper proof seal of the separated fittingprotects the medical waste personnel from coming in contact with any ofthe waste in the sealed waste container.

In an alternate embodiment the detachable fitting on the waste containermay not have any residual piece on the main instrument fitting butrather “breaks” or “snaps” away form the detachable piece on thedisposable waste container cleanly.

In an alternate embodiment, the waste module 6 could comprise two ormore waste containers wherein it is possible to remove one full wastecontainer while retaining one or more other waste containers attached toreceive waste from the working reaction modules. The microprocessorcould alert the technician that a waste container is in need ofreplacing by a sensor located in the waste container. If the technicianchooses to ignore the alert from the instrument, it could divert thewaste to another waste container until the time is convenient to replacethe full waste container. Since the staining apparatus operates each setof reaction components independently, the waste containers are set-up toreceive waste from any one or more of the reaction components duringoperation thereby eliminating the need to stop operation of theinstrument just to change any full waste container. The waste containerscan be hooked up in a series or in parallel, as explained above) to keepat least one waste container active while any other waste container isbeing changed. The microprocessor is preferably in direct communicationwith all the waste containers and will shut down any waste route thatleads to a fitting that has been detached and is in the process ofreplacement, repair, or cleaning.

In an alternate embodiment, the staining apparatus could have one mainwaste container which when full would alert the technician to start thewaste recovery procedure. The main waste container could be drained to asecondary waste container to be disposed. The waste container can becharged with activated charcoal or other neutralizing chemicals to aidin decontamination. The waste container can have a vent that has aneutralizing filter to release the build up of pressured vapors.

Turning again to the figures, it will be shown in greater detail how thesets of reaction components 104 (and others described herein) operate.

As explained above, an exemplary operation sequence of the reagent pack106 with the sets of reaction components 104 is generally shown in FIGS.7-22B.

The microscope slide 140 is loaded onto the heating element 136 of theslide support element 114 and positioned by location clips 138 or guidepegs or other orientation elements to verify proper location of themicroscope slide 140 on the slide support element 114. The slide supportelement 114 and microscope slide 140 is then moved into the reactioncompartment 112 wherein it is sealed via the O-rings 144 and 145 (orother sealing means contemplated herein). The reagent pack 106 is placedonto the reagent pack support device 116. The protocol is entered eitherautomatically or manually (described elsewhere herein) and the apparatusor staining apparatus 100 with the plurality of reaction components 104is instructed to start. Depending on the protocol the heating element136 can start to heat the microscope slide 140 or the protocol instructsthe dispensing of a reagent from the reagent pack 106 or from anothersource (e.g., a remote bulk source or X-Y-Z positioning device asdiscussed elsewhere herein) via the dispensing plunger 154.

If an individual reagent container 107 located on the reagent pack 106is selected, that particular reagent container 107 will be positionedover the injector port orifice 124 (over the microscope slide 140outside of the reaction compartment 112), and the dispensing plunger 154and depresses the piston within the reagent container 107 to expel thereagent 38 therefrom onto the microscope slide 140. The reagent pack 106would then be moved to position the rinse port aperture 108 in thereagent pack 106 (e.g., generally located between adjacent reagentcontainers 107) over the injector port orifice 124 wherein thedispensing plunger 154 would be lowered to seal the injector portorifice 124 or, additional air or reagent could be injected into thereaction compartment 112. Once the reagent 158 which has been applied tothe microscope slide 140 is removed from the microscope slide 140 bytilting the microscope slide 140 or by rinsing, the microscope slide 140can be further rinsed with a reagent or treated with pressurized airfrom the dispensing plunger 154.

As discussed elsewhere herein, the reaction compartments of the presentinvention can be pressurized (positively or negatively) during heatingof the reaction compartment or can be pressurized without heating, orpre-pressurized (positively or negatively) before the microscope slideor other reaction component is heated. The reaction compartment can bepre-pressurized, then heated, then repressurized to maintain a preferredpressure level within the reaction compartment. The reaction compartmentcan be pressurized either by vapor, gas, or steam produced by a reagent,solution, or liquid within the reaction compartment or by air, steam,inert gases, N₂ or any other gas typically used for pressurizingvessels, which is provided from an external source and is supplied viaair/pressure ducts or conduits or vacuum lines into the reactioncompartment, or by any other method described herein, such as by in situpressurization.

Embodiments of FIGS. 23-28

As shown in FIGS. 23-28 in an alternate version of the presentinvention, a staining apparatus 200 contains reaction components 204 aresimilar to reaction components 104 in comprising a reaction compartment212 similar to reaction compartment 112, a slide support element 214similar to slide support element 114, and a reagent pack support device216 similar to reagent pack support device 116. Reaction compartment 212comprises a reaction compartment heater 218 for heating the reactioncompartment 212 and optionally the slide support element 214 whendisposed therein or other gases or liquids therein. The reactioncompartment heater 218 has leads 220 thereto for connecting to anelectric power source (not shown). The reaction compartment 212 furthercomprises a reagent conduit 222 and an injector port orifice 224 fordelivering a reagent or other solution into the reaction compartment212. The reaction components 204 further comprise a reagent strip heater226 incorporated into the reagent pack support device 216 for heating areagent pack 206 (such as any of the reagent packs disclosed herein)disposed thereon. Leads 228 connect the reagent strip heater 226 to anelectric power source (not shown). The reaction compartment 212 furthercomprises a reagent conduit heater 230 for heating the reagent conduit222 thereby functioning to heat a reagent as it passes through thereagent conduit 222 into the reaction compartment 212 or merely onto amicroscope slide 250 if the reagent is applied when the microscope slide250 is outside of the reaction compartment 212. Leads 232 connect thereagent conduit heater 230 to an electric power source (not shown). Theslide support element 214 comprises a base 240 and, a handle 242, and afront O-ring 244 and a rear O-ring 246 for sealing the base 240 andmicroscope slide 250 within the reaction compartment 212. The slidesupport element 214 further comprises a microscope slide platform/heater248 and in operation has the microscope slide 250 disposed thereon, themicroscope slide 250 having an upper surface 251. The base 240 furthercomprises a base cavity 252 positioned below the slide platform/heater248 and has a base cavity heater 254 positioned therein and connectedvia lead 256 to an electric power source (not shown). The base cavityheater 254 functions to heat a reagent 258 disposed within the basecavity 252 to a temperature sufficient to heat the microscope slide 250and biological specimen and reagent 258 disposed thereon as describedelsewhere herein for other embodiments of the invention. The reagent 258in one preferred embodiment completely immerses the microscope slide 250as shown in FIG. 23. The reagent pack support device 216 in thisembodiment comprises a slot 260 (which may also be included in thereagent pack support device 116) therein for enabling a dispenserplunger (i.e., dispenser element) 264 to deliver a reagent 262 directlyupon the microscope slide 250 either when it is positioned within thereaction compartment 212 (FIGS. 23, 26, 27) or outside of the reactioncompartment (FIGS. 24, 25). As shown in FIGS. 24, 25, and 28 reagentsmay be applied to or removed from the microscope slide 250 when themicroscope 250 slide is positioned outside of the reaction compartment212 on the slide support element 214 and potentially outside of thestaining apparatus 200. Reagent may be removed from the microscope slide250 by the dispenser plunger 264 by moving the tip 266 of the dispenserplunger 264 over the microscope slide 250 and aspirating the reagenttherefrom. Reagent may be delivered to or removed from the microscopeslide 250 through one or more conduits 268 in the dispenser plunger 264(FIG. 28). The conduits 268 may function to provide reagents orsolutions, to remove reagents (via aspiration for example), or mayprovide air, gases, or liquids under pressure.

In other embodiments, reaction components 204 of the present inventionmay have any one or any combination of slide heating elements 136 or248, reaction compartment heater 218, reagent strip heater 226, reagentconduit heater 230, and base cavity heater 254, and when present any ofthe heating systems described herein may function individually andindependently of one another. The slide support element 214 may furtheroptionally comprise one or more drainage and/or supply conduits whichlead to the base cavity 252 for supplying the base cavity 252 with aliquid or other solution and for draining used liquid from the basecavity 252 after its use (e.g., by aspiration). Other supply ports,conduits, and ducts may supply the reaction compartments of the presentinvention such as are described in U.S. Pat. Nos. 6,534,008 and6,855,292.

In a preferred embodiment, the reaction compartment and/or slide supportelement of the present invention may be exposed to sterilizationconditions which may include high heat (e.g., above 100° C., or morepreferably above 130° C., and may use steam and/or chemicals to remove,or denature pathogens or residual chemicals or materials such as nucleicacids, antibodies, toxins or other proteins which remain in the reactioncompartment and slide support element after the reaction components areused. In a preferred embodiment, the reaction compartment and/or slidesupport element after heating is quickly cooled to near room temperatureor to below 50° C. within 3 sec, 5 sec, 10 sec or 20 sec for example tofurther denature or inactivate residual proteins or substances.

Further, although the various reaction components are shown herein ascomponents in discrete embodiments, it is contemplated that variouscomponents described herein can be assembled in any combination whichfunctions in accordance with the present invention.

Embodiments of FIGS. 29A-29F

Shown in FIGS. 29A-29F is a staining apparatus 300 which is at least oneof one or more of such chambers of a microscope slide staining apparatusof the present invention. The staining apparatus 300 has an inner space302, a front wall 304, a slide support element 310 having a heatingelement 312, and an optional handle 314, a reaction compartment 316,such as other reaction compartments described herein, a reagent packsupport device 318, a reagent plunger 319, and a reagent dispenser 320each of which is movable upwardly and downwardly in direction 321 andwhich may be movable laterally as well. When the slide support element310 is inserted into the inner space 302, an end portion of the slidesupport element 310 is preferably aligned flush with an outer surface ofthe front wall 304 as shown in FIGS. 29B-29E.

The slide support element 310 is similar to other slide support elementsdescribed herein and has sealing means 322 such as described elsewhereherein for enabling a microscope slide 324 to be sealingly enclosed onthe slide support element 310 within the reaction compartment 316. Areagent pack 42 (such as any reagent pack contemplated herein) can beinserted through an opening in the front wall 304 into the inner space302 of the staining apparatus 300 where it is secured on the reagentpack support device 318 for dispensing a reagent 328 onto the microscopeslide 324 via the reagent plunger 319 or by reagent dispenser 320 in amanner similar to that described for other such dispensers and plungersdiscussed elsewhere herein (in an alternate embodiment, the reagent packsupport device 318 (or any support devices contemplated herein) may beejected through an opening in the front wall 304 such that the reagentpack 42 can be loaded outside thereon of the staining apparatus 300.Shown in FIG. 29A the microscope slide 324 is initially in a placementposition outside of the reaction compartment 316 and inner space 302 ofthe staining apparatus 300. A microscope slide 324 is positioned on theheating element 312 of the slide support element 310 which is retractedin direction 326 into the reaction compartment 316 (FIG. 29B). A reagent328 is delivered to the microscope slide 324 via the reagent plunger 319(from the reagent pack 42) on the reagent pack support device 318 orfrom a remote reagent source via reagent dispenser 320 after thereaction compartment 316 has been retracted from the slide supportelement 310 in direction 326 (FIG. 29C). After the reagent 328 has beenapplied to the microscope slide 324, the reaction compartment 316 ismoved in direction 330 back over the slide support element 310 whereinthe sealing means 322 causes the slide support element 310 to be sealedagainst the inner surface 334 of the reaction compartment 316 so themicroscope slide 324 is sealed therein (FIG. 29D) within a pressurizabletreatment space 332 within the reaction compartment 316. Thepressurizable treatment space 332 is then pressurized via pressurizingmeans as described elsewhere herein (FIG. 29E). The microscope slide 324is heated by the heating element 312 to a predetermined temperaturewhich causes the reagent 328 on the microscope slide 324 to be heated toan elevated temperature above that which could be obtained absent theelevated pressure of the pressurizable treatment space 332. The heatedreagent 328 causes the desired biochemical/physical reaction within thebiological sample on the microscope slide 324 within the pressurizabletreatment space 332. After the reaction is completed, the pressure levelwithin the pressurizable treatment space 332 of the reaction compartment316 is returned to a normal (pre-pressurization) level and the reagent328 is removed therefrom by means such as those discussed elsewhereherein. The slide support element 310 is then ejected from the reactioncompartment 316 and/or inner space 302 of the staining apparatus 300 viadirection 330 wherein the microscope slide 324 can be removed therefrom(FIG. 29F). This can occur immediately after the heating step, or afterone or more additional steps or procedures has been performed on themicroscope slide 324. For example the steps of FIGS. 29B-29D (andoptionally FIG. 29E) can occur several times before the microscope slide324 is removed in the step of FIG. 29F.

Embodiments of FIGS. 30A-30F

Shown in FIGS. 30A-30F is a staining apparatus 340 which is at least oneof one or more of such chambers of a microscope slide staining apparatusof the present invention. The staining apparatus 340 has an inner space342, a front wall 344, a slide support element door 346 (shown open), areagent pack door 348, a slide support element 350 having a heatingelement 352, an optional handle 354, a reaction compartment 356, such asother reaction compartments described herein except having a closed end364, a reagent pack support device 366, and a reagent dispenser plunger368.

Reaction compartment 356 further comprises an inner space 362, apressure equalization conduit 358 between a forward portion of the innerspace 362 and a rear portion of the inner space 362 and a rear portionof the inner space 362. A conduit valve 360 is present in the pressureequalization conduit 358 for opening and closing the conduit 358 whendesired or for preventing backflow.

The slide support element 350 is similar to other slide support elementsdescribed herein such as shown in FIGS. 29A-29F, and has sealing means370 such as described elsewhere herein for enabling a microscope slide324 to be sealingly enclosed within the reaction compartment 356. Areagent pack 42 can be inserted through the opened reagent pack door 348into the inner space 342 of the staining apparatus 340 where it issecured on the reagent pack support device 366 for dispensing a reagent328 onto the microscope slide 324 via the reagent plunger 367 or via areagent dispenser 368 in a manner similar to that described for othersuch dispensers or plungers discussed elsewhere herein. In thisembodiment, the microscope slide 324 is initially in a placementposition outside of the reaction compartment 356 and the inner space 342of staining apparatus 340. A microscope slide 324 is positioned on theheating element 352 of the slide support element 350 which is retractedin direction 372 into the reaction compartment 356 (FIG. 30B). A reagent328 is delivered to the microscope slide 324 from the reagent plunger367 (from the reagent pack 42) on the reagent pack support device 366 orfrom a remote reagent source via reagent dispenser 368 after thereaction compartment 356 has been retracted from the slide supportelement 350 in direction 372 (FIG. 30C). After the reagent 328 has beenapplied to the microscope slide 324, the reaction compartment 356 ismoved in direction 376 back over the slide support element 350 whereinthe sealing means 370 causes the slide support element 350 to be sealedagainst the inner surface 357 of the reaction compartment 356 so themicroscope slide 324 is sealed therein within a pressurizable treatmentspace 378 within the reaction compartment 356. The pressurizabletreatment space 378 (also referred to herein a pressurization treatmentspace 378) is then pressurized (FIG. 30D) via “in situ pressurization”as explained below.

The microscope slide 324 is heated by the heating element 352 to apredetermined temperature which causes the reagent 328 on the microscopeslide 324 to be heated to an elevated temperature above that which couldbe obtained absent the elevated pressure in the pressurizable treatmentspace 378. The heated reagent 328 causes the desiredbiochemical/physical reaction within the biological sample on themicroscope slide 324 within the pressurizable treatment space 378. Afterthe reaction is completed, the pressure level within the pressurizabletreatment space 378 and head space 380 of the reaction compartment 356is returned to normal and the reagent 328 is removed therefrom by meanssuch as those discussed elsewhere herein. The slide support element 350is then ejected from the reaction compartment 356 and/or inner space 342of the staining apparatus 340 via direction 384 wherein the microscopeslide 324 can be removed therefrom (FIG. 30F). This can occurimmediately after the heating step, or after one or more additionalsteps or procedures has been performed on the microscope slide 324. Forexample the steps of FIGS. 30B-30E can occur several times before themicroscope slide 324 is removed in the step of FIG. 30F.

In a preferred embodiment of the reaction compartment 356 (and of otherreaction compartments contemplated herein), the sealing means 370comprises a ground or polished glass seal in a surface portion of thereaction compartment 356 which can hold pressure from a separate bulksource of pressure to pressurize the pressurization space 378 of thereaction compartment 356 or, in an alternative embodiment this polishedseal (or other seals described herein) can also produce and holdpressure inside the pressurization space 378 of the reaction compartment356 without the need for a separate bulk pressure source being sent toeach reaction compartment 356.

This method of pressure generation, operationally represented in FIG.30E, is referred to herein as “in-situ pressurization”. The veryeffective sealing means 370 of the present invention can form apressurization treatment space which is sufficiently sealed to produceand/or increase and/or decrease the pressure of atmospheric pressureconditions inside the pressurization treatment space 378 of the reactioncompartment 356. After the slide support element 350 and slide thereonis sealed within the reaction compartment 356 the slide support element350 is moved further into the inner space 362 of the reactioncompartment 356 to produce pressure therein by forcing the trappedresidual atmospheric air in the pressurization space 378 surrounding themicroscope slide 324 in the reaction compartment 356 and inside a headspace 380 of the reaction compartment 356. For example, in oneembodiment the reaction compartment 356 (e.g., having a length of 8inches can have the slide support element 350 inside the first 5 inchesof its length. The head space 380 comprises the remaining 3 inches ofspace within the reaction compartment 356. The reagent 328 has beenadded to the microscope slide 324 present on the slide support element350. The slide support element 350 is then moved farther into the headspace 380 of the reaction compartment 356, for example 0.01 to nearly 3inches. This movement, further into the reaction compartment 356 causesthe gas (e.g., air) in the head space 380 between the closed end 364 ofthe reaction compartment 356 and a distal end 351 of the slide supportelement 350 to compress. This compression of the air in the head space380 produces pressure above the original pressure in the reactioncompartment 356. This pressure is diverted to the pressurizationtreatment space 378 by conduit 358 through valve 360. The head space 380is only in contact with the pressurization treatment space 378 about themicroscope slide 324, and vice versa, by the conduit 358 or other meansto connect the head space 380 with the pressurization treatment space378.

This connection with the head space 380 to the pressurization treatmentspace 378 may include as noted a one-way or two-way conduit valve 360 orother means of transferring the pressure in the compressed head space380 to the pressurization treatment space 378 without allowing thecontents of the pressurization treatment space 378 to be communicated ormoved toward or into the head space 380 for possible contamination ofthe head space 380 or vice versa.

The pressurization conduit 350 is shown in FIGS. 30A-30F as a conduitbetween proximal and distal portions of the reaction compartment 356,however the conduit may instead be wholly within a distal portion of theslide support element 350 (e.g., see FIGS. 33A-33H).

Although the pressured gas or air produced from the compressed headspace 380 can move into the pressurization treatment space 378 there isa need to stop any contamination of the gas or air in the compressedhead space 380 with the contents of the pressurization treatment space378 and vice versa. Valves or other systems known in the art can be usedto inhibit or stop this potential backflow and/or cross-contamination.These conduit valves 360 can be, but are not limited to, in line wateror gas dedicators, one-way valves, two-way valves, a one way pressureopening valves, metered ports, or any other device able to be used toprevent the contents from one compartment or area being contaminatedwith the contents of another compartment or area.

The amount of pressure in the pressurization treatment space 378 isproportional to the degree of movement of the slide support element 350into the head space 380 of the reaction compartment 356. The pressureproduced is directly related to the length and outer diameter of theslice support element 350 and the length and inner diameter of thereaction compartment 356 along with the total travel length of the slidesupport element 350 or the reaction compartment 356 with the movement tocompress the head space 380 with a normal atmospheric pressure trapbetween the front of the reaction compartment 356 and the closed end 364of the reaction compartment 356 to produce the increased pressure bycompressing the air or gas trapped in the head space 380. The pressurein the head space 380 for example could be 20 psig caused by compressingthe residual air trapped in the head space 380 and the now pressurizedair could be delivered via the conduit 358 to the pressurized treatmentspace 378 containing the microscope slide thereby equilibrating thepressure of the pressurized head space with the pressure in thepressurized treatment space 378. Evaporation of reagents associated withthe biological specimen, under heat could also contribute to thepressure in the pressurized treatment space 378. As noted above, aconduit valve 360 can be present for preventing contents of thepressurization treatment space 378 from moving into the head space 380through the conduit 358. The pressure in the head space 380 can beincreased or decreased before, during, or after the heating element 352heats the reagent 328 in contact with the microscope slide 324. Sincethe reaction compartment 356 may have a heating device in its walls, inone embodiment of the invention, a liquid could also be added to thehead space 380 to produce steam or gas to be sent through the conduit358 to pressurize the pressurization treatment space 378 of the slidesupport element 350.

In summary, the head space 380 can be used to cause pressurization ofthe pressurization treatment space 378 (above or below atmosphericpressure) before, during, or after the heating elements 352 are turnedon without having an external source of pressure used to pressurize thepressurization treatment space 378. Further, the presently described insitu pressurization of the pressurization treatment space 378 can occurwithout use of heat from heating elements. Alternatively, as noted,liquid could be added to the head space 380 to induce pressurization bysteam or vapors or add further pressure to the pressurization treatmentspace 378 in the reaction compartment 356 whether the head space 380 iscompressed or not. This apparatus could also be able to draw a vacuuminto the pressurization treatment space 378, for example by reversingthe movement of the slide support element 350 and pulling the reactioncompartment 356, the slide support element 350, or both, in oppositedirections to produce a vacuum in the conduit 358 and thereby placingthe vacuum in both the head space 380 and pressurization treatment space378. This method can also be used to regulate the pressure inside thepressurization treatment space 378 regardless of the source of thepressure by moving the reaction compartment 358 and slide supportelement 350 together or separately to cause a pressure or vacuumenvironment to regulate the pressure or vacuum conditions within thereaction compartment 356. In one example, if the pressure is desired tobe maintained at 30 psig in the pressurization treatment space 378, theregulation can come from the pressurized or depressurized head space380. This regulation is available no matter how the pressure was or isoriginally being maintained. For example, if the microprocessor sensesthe pressure in the pressurization treatment space 378 exceeds thedesired temperature or is too low, the position of the slide supportelement 350 or reaction compartment 356 could be adjusted slightly tochange the pressure level. Or the microprocessor could use this headspace pressure regulation process to quickly reduce or add pressure tothe pressurization treatment space 378 for a condition that might becomedangerous to the limits of the strength and integrity of the reactioncompartment 356 as a failsafe option. The change in pressure in the headspace 380 can be a fine adjustment or coarse adjustment to the pressurein the pressurization treatment space 378. The adjustment increments canbe of any measurable amount. The adjustment can be as little as 0.001psig above or below atmospheric pressure. Preferably the adjustment isin 0.5 psig increments either above or below atmospheric pressure.

In this embodiment of the present invention, as noted “in-situ”pressurization and vacuum (above atmospheric or below atmosphericpressure) is caused by compressing the head space 380 in the portion ofthe reaction compartment 356 between the closed end 364 thereof and thedistal end 351 of the slide support element 350. An individual reactioncompartment 356 can move in relation to the slide support element 350 orthe slide support element 350 can move in relation to the correspondingindividual reaction compartment 356. The individual reaction compartment356 and the slide support element 350 can move independently of eachother and/or simultaneously with each other to compress the head space380 present between the individual reaction compartment 356 and thedistal end 351 of the slide support element 350. In the preferredembodiment of the “in-situ” production of pressure described herein, inwhich a single individual reaction compartment 356 is sealed about asingle slide support element 350, both are independently movable inrelation to each other under pressure and wherein pressure is producedby the relative movement of each other and the sealed head space 380 inrelation to the sealed individual reaction compartment 356 and thesingle slide support element 350. As noted previously, the reactioncompartment 356 can be modified to hold more than one microscope slideper slide support element (e.g., 2 or more) if desired and still be ableto produce “in-situ pressurization”. A reaction compartment 356 could besized to hold multiple slide support elements 350 moving on a singleplatform that can be joined with a reaction compartment 356 which iscomplementary with the larger slide support platform. In situpressurization via compression of the head space 380 of the reactioncompartment 356, can be performed without addition of additionalpressurization from a remote pressurization means thus reducing thecomplications inherent in using such a remote source for example therequirement of tubes, valves, and conduits able to tolerateabove-atmospheric or below-atmospheric pressures.

Embodiments of FIGS. 31A-31F and 32

Shown in FIGS. 31A-31F and 32 is a staining apparatus 300 a which is atleast one of one or more of such chambers of a microscope slide stainingapparatus of the present invention. The staining apparatus 300 a has aninner space 302 a, a front wall 304 a, a slide support element door 306a (shown open), a reagent pack door 308 a, a slide support element 310 ahaving a heating element 312 a, an optional handle 314 a, a reactioncompartment 316 a, a reagent pack support device 318 a, a reagentplunger device 319 a and a reagent dispenser 320 a.

The staining apparatus 300 a is substantially the same as stainingapparatus 300 of FIGS. 29A-29F except that the reaction compartment 316a differs from reaction compartment 316 of FIG. 29A in that it has anopen portion or “window” 317 through which the reagent 328 can beapplied to the microscope slide 324 from or by the reagent plunger 319 aor via the reagent dispenser 320 a. In this embodiment with “windows”,the window 317 is advantageous in enabling the reagent to be dispensedupon the microscope slide 324 without having to be passed through anarrow reagent conduit. Further, a dispenser element associated with theX-Y-Z positioning device (e.g. such as reagent dispenser 320 a) does nothave to be adapted for use with a reagent pack to be able to be usedwith the reaction compartment window 317.

The reaction compartment 316 a can be rotated about the slide supportelement 310 a to close the window 317 to form a pressurizable treatmentspace 332 a around the microscope slide 324 in the reaction compartment316 a. In this embodiment, preferably, the sealing means 322 a is aground and polished glass surface that can be easily rotated to open andclose the window 317. Reaction compartment 316 a with window 317 isshown in a perspective view in FIG. 32. The rotational movement in thisembodiment of the reaction compartment 316 a can be a few degrees or canbe 180° or more in relation to the microscope slide 324. Thus, thewindow 317 of the reaction compartment 316 a can be positioned directlyabove the microscope slide 324 (in a 0° position or “open” position) orcan be rotatingly moved through a range of positions to be directlyunder the microscope slide 324 (180° position or “closed” position),rotating in either direction from the 0° home (open) position to theclosed position wherein the window is covered by a lower surface of theslide support element 310 a. The sealing means 322 a can be of any typeknown in the art of sealing pressurized vessels. The preferred sealingmeans 322 a is a ground and polished glass seal. This type of seal isknown in the art of ground and polished seals for glass hypodermicsyringes for example which are manufactured and sold under the tradename Micro-Mate® by Popper and Sons, Inc. New Hyde Park, NY, and thussuch ground and polished glass seals are known in the art.

The slide support element 310 a is similar to other slide supportelements described herein and has sealing means 322 a such as describedelsewhere herein for enabling the microscope slide 324 to be sealinglyenclosed within the reaction compartment 316 a. A reagent pack 42 can beinserted through the opened reagent pack door 308 a into the inner space302 a of the staining apparatus 300 a where it is secured on the reagentpack support device 318 a (or otherwise positioned thereon) fordispensing a reagent 328 a onto the microscope slide 324 via the reagentplunger 319 a or via reagent dispenser 320 a in a manner similar to thatdescribed for other such dispensers or plungers discussed elsewhereherein except that the reagent 328 is preferably disposed through thewindow 317 of the reaction compartment 316 a. In this embodiment, themicroscope slide 324 is initially in a placement position outside of thereaction compartment 316 a and inner space 302 a of the stainingapparatus 300 a. The microscope slide 324 is positioned on the heatingelement 312 a of the slide support element 310 a which is retracted indirection 326 a into the reaction compartment 316 a (FIG. 31 B). Areagent 328 is delivered to the microscope slide 324 from the reagentplunger 319 a (from the reagent pack 42) on the reagent pack supportdevice 318 a or from a remote reagent source via reagent dispenser 320 a(FIG. 31C). Then the reaction compartment 316 a is rotated 180° (orother equally effective amount) about the slide support element 310 a indirection 329 (FIG. 31D), thereby closing the window 317 wherein thesealing means 322 a causes the slide support element 310 a to be sealedagainst an inner surface 334 a of the reaction compartment 316 a so themicroscope slide 324 is sealed therein within the pressurizationtreatment space 332 a. The pressurization treatment space 332 a is thenpressurized (FIG. 31D) via pressurizing means as described elsewhereherein. The microscope slide 324 is then heated by the heating element312 a to a predetermined temperature which causes the reagent 328 on themicroscope slide 324 to be heated to an elevated temperature above thatwhich could be obtained absent the elevated pressure in thepressurization treatment space 332 a. The heated reagent 328 causes thedesired biochemical/physical reaction within the biological sample onthe microscope slide 324 within the pressurization treatment space 332a. After the reaction is completed, the pressure level within thepressurization treatment space 332 a of the reaction compartment 316 ais returned to normal, the reaction compartment 316 a is rotated to thehome (open) position (FIG. 31E), and the reagent 328 is removedtherefrom by means such as those discussed elsewhere herein. The slidesupport element 310 a is then ejected from the reaction compartment 316and/or inner space 302 a of the staining apparatus 300 a via movement indirection 330 wherein the microscope slide 324 can be removed therefrom(FIG. 31F). This can occur immediately after the heating step, or afterone or more additional steps or procedures has been performed on themicroscope slide 324. For example the steps of FIGS. 31C-31E can occurseveral times before the microscope slide 324 is removed in the step ofFIG. 31F.

Embodiments of FIGS. 33A-33H

Shown in FIGS. 33A-33H is a staining apparatus 340 a which is at leastone of one or more of such chambers of a microscope slide stainingapparatus. The staining apparatus 340 a has an inner space 342 a, afront wall 344 a, a slide support element door 346 a (shown open), areagent pack door 348 a, a slide support element 350 a having a heatingelement 352 a, a distal end 351 a, an optional handle 354 a a reactioncompartment 356 a, which combines the elements of other reactioncompartments described herein such as reaction compartments 316 a and356, a reagent pack support device 366 a, a reagent plunger 367 a, and areagent dispenser 368 a.

In particular, the reaction compartment 356 a has a closed end 364 a,and inner surface 357 a, an inner space 362 a, and a window 365 throughwhich a reagent 328 can be applied in the manner shown in FIGS. 31A-31F.The slide support element 350 a comprises a pressure equalizationconduit 358 a which is similar to the pressure equalization conduit 358of FIGS. 30A-F in that the conduit 358 allows pressure equalizationbetween a forward portion of the inner space 362 a (which constitutes apressurization treatment space 378 a where the microscope slide 324 ispositioned) and a rear portion which constitutes a head space 380 a ofthe reaction compartment 356 a, but which is different therefrom in thatconduit 358 a is positioned in a distal portion 351 a of slide supportelement 310 a rather than in reaction compartment 356 a.

The slide support element 350 a is similar to other slide supportelements described herein and has sealing means 370 a such as describedelsewhere herein for enabling a microscope slide 324 to be sealinglyenclosed within the reaction compartment 356 a. A reagent pack (notshown) can be inserted through the opened reagent pack door 346 a intothe inner space 342 a of the staining apparatus 340 a where it issecured on the reagent pack support device 366 a for dispensing areagent 328 onto the microscope slide 324 via the reagent plunger 367 aor via reagent dispenser 368 a in a manner similar to that described forother such dispensers and plungers discussed elsewhere herein. In thisembodiment, the slide support element 350 a is initially in a placementposition outside of the reaction compartment 356 a and stainingapparatus inner space 342 a (FIG. 33A). A microscope slide 324 ispositioned on the heating element 352 a of the slide support element 350a which is retracted in direction 372 into the reaction compartment 356a (FIG. 33B). A reagent 328 is delivered through window 365 to themicroscope slide 324 from the reagent plunger 367 a (from the reagentpack (not shown)) on the reagent pack support device 366 a or from aremote reagent source via reagent dispenser 368 a after the reactioncompartment 356 a has been retracted from the slide support element 350a in direction 372 (FIG. 33C). After the reagent 328 has been applied tothe microscope slide 324, the reaction compartment 356 a is rotated 180°(or other appropriate amount) in direction 373 wherein the sealing means370 a causes the slide support element 350 a to be sealed against theinner surface 357 a of the reaction compartment 356 a in the same manneras in FIG. 31D wherein the microscope slide 324 is sealed therein (FIG.33D) within a pressurization treatment space 378 a. The pressurizationtreatment space 378 a is then pressurized as shown in FIGS. 33E-33F inthe same “in situ pressurization” method shown and described in regardto FIGS. 30D-30E. The microscope slide 324 in the pressurizationtreatment space 378 a is heated by the heating element 352 a to apredetermined temperature which causes the reagent 328 on the microscopeslide 324 to be heated to an elevated temperature above that which couldbe obtained absent the elevated pressure in the pressurization treatmentspace 378 a. The heated reagent 328 causes the desiredbiochemical/physical reaction within the biological sample on themicroscope slide 324 within the pressurization treatment space 378 a(FIG. 33F). After the reaction is completed, the pressure level withinthe pressurization treatment space 378 a of the reaction compartment 356a is returned to normal (FIG. 33G) and the reagent 328 is removedtherefrom by means such as those discussed elsewhere herein. Additionalreagent can then be applied to the slide through the window 365 ifdesired. The slide support element 350 a is then ejected from thereaction compartment 356 a and/or inner space 342 a of the stainingapparatus 340 a via direction 384 wherein the microscope slide 324 canbe removed therefrom (FIG. 33H). This can occur immediately after theheating step, or after one or more additional steps or procedures hasbeen performed on the microscope slide 324. For example the steps ofFIGS. 33C-33G can occur several times before the microscope slide 324 isremoved in the step of FIG. 33H.

Shown in FIG. 34 an alternate embodiment of the invention is representedas the staining apparatus 400. The staining apparatus 400 has a frontwall 402, a back wall 404, a first side wall 406, a second side wall408, and an inner space 410. Inside the staining apparatus 400 are aplurality of sets of reaction components 412 (six are shown but more orless may be included) similar to the reaction components of any of 1-6and 29A-33H. In particular, each set of reaction components 412comprises a movable reaction compartment 414 and a movable slide supportelement 416 each which is independently movable of each other reactioncompartment and slide support element respectively. The slide supportelement 416 is moved forwardly, backwardly, and rotatingly by a motorassembly 418 comprising a motor 420 and a shaft 422. A motor assemblyfor moving the reaction compartment 412 forwardly, backwardly andpreferably rotatingly is not shown. The staining apparatus 400 isoperable in any of the configurations represented in FIGS. 1-6 and29A-33H and as contemplated elsewhere herein and further as describedherein. For example, slide support elements 416 can be moved into andout of the inner space 410 of the staining apparatus 400, and into andout of reaction compartments 412; similarly, reaction compartments 412can be moved over and sealed about slide support elements 416 orretracted to expose the slide support elements 416. Staining apparatus400 is further shown as having an X-Y-Z positioning device 430 having asa movable head 432 discussed elsewhere herein which is positioned in theinner space 410 such that the movable head 432 can be moved laterallyand vertically over the slide support elements 416 on a rail 434. Themovable head 432 in one embodiment of the X-Y-Z positioning device 430comprises a dispensing element for dispensing a reagent or otherdispensable material, such as a cover slip or a bonding material forattaching a cover slip. The movable head 432 may comprise a bar codereader or other mechanism for obtaining information from the microscopeslide or from the reagent pack. The movable head 432 may comprise aninkjet printer or laser etching device or other light emitting devicefor imparting or printing a pattern, symbol, or label on the microscopeslide or other device described herein. The movable head 432 maycomprise an aspirator for removing a reagent or solution from themicroscope slide or slide support element. The staining apparatus 400may comprise multiple X-Y-Z positioning devices 430 and/or multiplemovable heads 432, each separate movable head 432 able to perform one ormore of the functions contemplated herein. For example, in onenon-limiting example, the staining apparatus 400 may comprise one X-Y-Zpositioning device 430 comprising a movable head 432 which is an inkjetprinter, another X-Y-Z positioning device 430 comprising a movable head432 which is an optical code reader and/or scanner, and another X-Y-Zpositioning device 430 comprising a movable head 432 which is reagentdispenser and/or reagent aspirator. In the staining apparatus 400, thereagents are applied to the microscope slide and/or slide supportelement 416 within the same chamber compartment that contains thereaction compartments 414. In one embodiment of the invention, theoptical scanner of the X-Y-Z positioning device may scan the microscopeslide on the slide support element to identify and record the locationof the biological (tissue) specimen thereon. This information can beused to optimize the placement of the reagent on the microscope slide sothat it is deposited directly upon the biological specimen or in apreferred location on the microscope slide for mixing or treatmentpurposes.

FIG. 35 shows a staining apparatus 400 a which is similar to stainingapparatus 400 except that staining apparatus 400 a comprises (1) apressurizable common chamber 446 wherein microscope slides onindependent slide support elements 416 a are exposed to the samepressurization level therein, and (2) a common application chamber(treatment chamber) 444 wherein reagents are applied to the microscopeslides. Microscope slides are first inserted into a non-pressurizedcommon application chamber 444 where a reagent is applied thereto by areagent pack, and/or an X-Y-Z positioning device 430 a. Afterapplication of the reagent to the microscope slide 446, the slidesupport element 416 a passes into the pressurizable common chamber whereeach slide support element 416 a is first sealed within thecorresponding reaction compartment 414 a also referred to herein as acorridor or enclosable compartment until the opening through which theslide support element 416 a passes into the pressurizable common chamber446 is closed or sealed. Once the slide support element 416 a has beensealed within the pressurizable common chamber 446, the reactioncompartment can be retracted to expose the microscope slide to thecommon pressure level established therein. The advantage of theembodiment of FIG. 35 is that there are fewer components necessary incomparison to the embodiment of FIG. 34, since in FIG. 34 each reactioncompartment 414 is separately pressurized, wherein in FIG. 35 each“reaction compartment” 414 a is not individually pressurized. Furtherexplanation is provided below.

In the pressurizable common chamber 446 of the present invention such asis shown in FIG. 35 the slide support elements 416 a can be moved intoand out of the pressurizable common chamber 446 while said chamber isunder pressure that is exceeding or is below atmospheric pressure whilesaid pressure is maintained in the pressurizable common chamber 446 evenwhen the independently moving side support elements 416 a are movinginto the pressurizable common chamber 446 to be treated or are beingmoved out of the pressurizable common chamber 446 for removal of thetreated slide or for placement of a new slide on the slide supportelement 416 a to be moved into the pressurized pressurizable commonchamber 446 for treatment under pressure. The slide support elements 416a can be moved into and out of the pressurizable common chamber 446without changing or releasing or diminishing the pressure therein. Themovement is such that each slide support element 416 a is moved througha corridor or reaction compartment 414 a that is sealed when thereaction compartment 414 a is sealed at seals 442 to the wall 440 whichseparates the pressurizable common chamber 446 from the applicationchamber (treatment chamber) 444 that isolates the slide support element416 from the inner space of the pressurizable common chamber 446. Theindividual corridor or reaction compartment 414 a is able to be sealedat its proximal end to seal the proximal end against the wall 440 of thepressurizable common chamber 446 having openings through which the slidesupport element can pass. This seal 442 can be any sealing meanscontemplated herein or any other sealing means able to function inaccordance with the invention. The individual independently moving slidesupport element 416 a within the corridor or enclosable compartment 414a could now move through an opening in the wall 440 of the pressurizablecommon chamber 446 while inside the sealed inner space of the sealedcompartment 414 a. Even after the slide support element 416 a has movedthrough the access opening of the pressurizable common chamber 446, theenclosable compartment 414 a or corridor remains sealed over the openingin the wall to maintain pressure within the chamber 446.

FIG. 36 shows a staining apparatus 400 b which is similar to stainingapparatus 400 a in that staining apparatus 400 a comprises (1) apressurizable common chamber 446 b wherein microscope slides onindependent slide support elements 416 b are exposed to the samepressurization level therein, and (2) a common application chamber(treatment chamber) 444 b wherein reagents are applied to the microscopeslides. Microscope slides are first inserted through a door or sealingmeans 403 b into the non-pressurized common application chamber 444 bwhere a reagent is applied thereto from a reagent pack, and/oroptionally an X-Y-Z positioning device (not shown) supplied from aremote source. After application of the reagent to the microscope slide446 b, the slide support element 416 b passes through a door 441 b intothe pressurizable common chamber 446 b where each slide support element416 b is sealed therein upon closure of the door (or sealing means 441b. Once all slide support elements 416 b have been sealed within thepressurizable common chamber 446 b, the common pressure level can beestablished therein and the treatment protocol can proceed. In theembodiment of FIG. 36 all microscope slides are exposed to the samepressure level.

Referring now to FIGS. 37A-39B, the present invention is furtherdirected to a novel method of applying (spreading) a reagent to amicroscope slide or analytic plate or substrate having a biologicalspecimen attached thereto. In one embodiment of the invention, thereagent is a DNA or RNA “probe” but may be any reagent described herein,including a liquid adhesive material. Such probes are well known in theart. Probes or “probe mixtures” (and other reagents) are expensive andit is an object of the present invention to provide a technique thatefficiently applies the reagent mixture to the microscope slide tooptimize coverage thereover yet which uses only a minimum amount of thereagent mixture. The amount of a reagent, such as a probe, that isroutinely used to perform manual in-situ hybridization is 10 μl under astandard 22 mm×22 mm cover slip. The present invention contemplatesutilization of the similar volume of reagent (e.g., 10 μl) but canevenly spread this quantity of probe mixture across a surface areagreater than 22 mm×22 mm.

Referring in particular to FIGS. 37A-37F, an example of such a spreadingdevice is shown. The spreading device 500 has a gap 502 that, forexample, is 3-25 μm deep (but may be deeper). Typically, a tissuespecimen (biological specimen) 504 used for in-situ hybridization isplaced on a microscope slide 506. The microscope slide 506 has a labelend 508 and a treatment surface 510. The thickness of the tissuespecimen 504 is typically between 2-7 μm and more preferably between 4-5μm. Thus the gap 502 of the spreading device 500 preferably has a depththat is 1-23 μm higher than the tissue specimen 504; or 2-15 μm, or 3-10μm, or 5-7 μm above the tissue specimen 504. The spreading device 500can be, for example, one inch wide and have end blocks 512 that are upto 1 inch in length and generally 0.01-5 μm in width. These end blocks512 thus touch the microscope slide 506 0.01-5 μm from the edge of themicroscope slide 506. The space between the two end blocks 512 and themicroscope slide 506 encompasses the gap 502 of the spreading device500. Preferably the depth of gap 502 extends at least 0.01 μm-50 μmabove the highest point of the tissue specimen 504 on the microscopeslide treatment surface 510. Preferably the depth of gap 502 is 0.1-5μm, or more preferably 1-3 μm above the tissue specimen 504 to becovered by the reagent 514 disposed thereon.

The depth of the gap 502 of the spreading device 500 determines thethickness of a layer 516 (also referred to herein as a film or coating)of the reagent 514 that can be spread across the microscope slide 506evenly. The thickness of the layer 516 is important so the reagent 514forms a film or coating that is distributed evenly across the treatmentsurface 510 and the tissue specimen 504 thereon with the predeterminedthickness of the gap 502 of the spreading device 500. The length of thespreading device 500 (measured from across the width of the slide 506)can be any size to accommodate the tissue specimen 504 on the slide 506.Tissue specimens 504 can be of any size in the art that can be placed,for example on a microscope slide 506 or other appropriate analyticplate. Even a very tiny tissue specimen 504 can have a thin coating ofreagent 514 spread across its surface by the spreading device 500 of thepresent invention. For example, the total length of the spreading device500 could be as little as 3-5 mm, wherein the length of the gap 502across the length of the spreading device 500 is generally about 1-4 mm.In this version, the width of the layer 516 would be 1-4 mm, and thethickness would be the depth of the gap of the spreading device 500. Inan alternate embodiment (FIG. 37B), a spreading device 500 a is likespreading device 500 except it comprises block portions 512 a whichextend about a portion of the underside of the slide 506. The spreadingdevice 500 a may have a handle 518 to enable it to be moved manually.The spreading device 500 may be moved across the microscope slide 506along a track 520 which may be operatively associated with a motor orother means of causing movement of the spreading device 500.

In one example, the tissue specimen 504 is a prostate or breast biopsysample which is 1 mm wide and 1.2 cm long. A very small spreading device500 as described above could be used to lay a thin layer 516 of reagent514 over the entire tissue specimen's width and length. The spreadingdevice 500 (or 500 a) of the above example could be about 3 mm wide andhave a gap depth of 6-7 μm in gap 502. A 2-4 μl drop of reagent 514could then be used to lay the thin layer 516 over the tissue specimen504 by movement of the spreading device 500 (or 500 a) thereover withoutany waste of reagent 516. The spreading device 500 (or 500 a) of thepresent invention can be of any size that is necessary to lay a thinlayer 516 of reagent 514 over a biological (tissue) specimen 504 on asubstrate such as a microscope slide 506. Other biological testingsubstrates are known and can be used with the present invention such asPetri dishes, plates of glass or plastic, and others as discussedelsewhere herein.

The spreading device of the present invention preferably has a gap 502preferably is at least 0.01-20 μm above the tissue specimen 504. Thethickness of the tissue specimen 504 is between 3-7 μm and morepreferably between 4-5 μm. The spreading device 500 (or 500 a) of thepresent invention may have a gap that is 4-10 μm and more preferablybetween 6-7 μm or just one, two, or three pm above the biologicalspecimen. The gap depth of the spreading device can be, for example,0.01 μm, to 0.1 μm, 0.1 μm to 1 μm, 1 μm to 20 μm, 20 to 50 μm above themicroscope slide's 506 surface. Preferably the thickness of gap 502 is1-3 μm above the specimen 504 to be covered by the reagent or solutionfilm. The thickness of the gap 502 determines the thickness of the layer516 or film of reagent 514 that can be spread across the slide 506evenly. The thickness is important so the reagent 514 forms a layer 516that is distributed evenly across the microscope slide 506 and specimen504 with a thickness of the gap of the spreading device 500 (or 500 a).The length of the spreading device 500 (or 500 a) can be any size toaccommodate a biological specimen. The reagent 514 that can be spread bythe spreading device 500 (or 500 a) can be any reagent used in alaboratory setting including, but not limited to: stains, probes, DNAand RNA molecular probes, immunoreagents, histochemical reagents,antibodies, in-situ reagents, mineral oils, ionic or non-ionic reagentsadditives, SDS, Tween, Brij, detergents, alcohols, polyols, glycols,de-waxing solutions, hydrating solutions, fixatives, detection reagents,thermoplastic resins, plastic polymers, cover slip mountants forcoverslipping the specimen without the need for plastic or glass coverslips, fixatives, etc.

When using the spreading device 500 (or 500 a) of the present inventionon a microscope slide 506, the initial position of the spreading device500 (or 500 a) could be at either terminal end of the treatment surface510 of the microscope slide 506. The distal end away from the label end508 of the microscope slide 500 (the non-label end) is a preferredinitial starting position (FIGS. 37C-37D). The reagent 514 is placed asa drop in front of the spreading device 500 (or device 500 a) (FIGS.37C-37D), then the spreading device 500 is moved over the drop ofreagent 514, over the tissue specimen 504, and to the label end 508 ofthe slide 506 thereby depositing the layer 516 of reagent 514 evenlyacross the slide (FIG. 37E). Once the spreading device 500 (or 500 a)has touched the drop of reagent 514, the reagent 514 spreads across thegap 502 of the spreading device 500 (or 500 a) by capillary action andthe spreading device 500 (or 500 a) is moved slowly toward the label end508 of the microscope slide 506. The end blocks 512 (or 512 a) passlengthwise over the peripheral side edges of the microscope slide 506.The reagent 514 is thus spread evenly under the gap 502 of the spreadingdevice 500 (or 500 a) across the microscope slide 506. The spreadingdevice 500 (or 500 a) is then retracted to the starting position on theslide (FIG. 37F). The thickness of the layer 516 of reagent 514deposited is dependent on the viscosity of the reagent 514 and the depthof gap 502 of the spreading device 500 (or 500 a). The viscosity of thereagent 514 can be of any viscosity known in reagents for laboratorytesting. In one example, the viscosity may be that of mineral oil atambient room temperature. Molecular probe dilutions have similarviscosity to mineral oil and this is a viscosity that can be used bythis method of the present invention.

The spreading device 500 (or 500 a) of the present invention can bedisposable or reusable. The spreading device of the present inventioncan be molded out of plastic, thermoplastics, polymers, metal, glass,ceramic, and/or rubber, or combinations thereof, and can be labeled orcolor-coded to indicate the thickness the gap of the spreading device.The spreading device may be constructed of metal and coated with apolymer or plastic. In one example, a spreading device may be rated ashaving a gap of 6 μm, and has that numerical number stamped thereon, andhas a particular color such as blue. This “blue” applicator when usedwould lay down a reagent layer with a thickness of up to 6 μm across themicroscope slide for example. In an alternate embodiment, the spreadingdevice can have a handle attached thereto for manual use (see FIG. 37B),or other appendages for the attachment to an automated instrumentdescribed in further detail below. The spreading device can spread alayer of a film or any reagent used in the laboratory setting such as,but not limited to: stains, probes, DNA and RNA molecular probes,immunoreagents, histochemical reagents, antibodies, detection reagents,thermoplastic resins and mountants for coverslipping the specimenwithout the need for plastic or glass cover slips, or fixatives.

As noted above for FIGS. 37-39, the spreading device is preferablyautomatically movable. The spreading device may comprise a plastic orpolymer coated metal gap applicator which can be moved by a movingmagnet present in the slide support element. The reagents used with thespreading device can have detergents present to help the spreading outof the reagents. These detergents are ionic or non-ionic detergents,glycols, polyols, etc.

As explained elsewhere herein, in one version of the method of using thespreading device 500, a microscope slide is placed on the slide supportelement, the correct spreading device is loaded onto the slide supportelement and rests on the slide, the microscope slide is moved into thestaining apparatus to the treatment and application position, a reagentis either dispensed by the reagent pack, X-Y-Z dispenser, dispensingelement, a remote source, or the reagent is dispensed from the dispenserintegrated into the spreading device, the spreading device moves acrossthe microscope slide and over the biological specimen to lay down anexact thickness of reagent equal to the thickness of the gap of thespreading device, the microscope slide is incubated and rinsed, andanother reagent then can be dispensed onto the slide or the dispensedreagent can be spread again by the spreading device until the protocolis complete. If the slide is to be coverslipped by the spreading devicethe final reagent would be applied to the dried microscope slide and acoverslip mountant would be applied to front of the spreading devicewhich would move across the slide to lay down an exact thickness ofcoverslip mountant to the slide. The slide is then heated to dry andharden the coverslip reagent and the slide is then removed and can godirectly to the microscope for evaluation by a technician.

In reference to FIGS. 38A, 38B, 39A, and 39B, the slide support elementsand associated reaction compartments contemplated herein (such as, butnot limited to, slide support element 310, and reaction compartment 316)can be modified to incorporate the spreading device 500 (or 500 a)described herein. The spreading device 500 for example, can be attachedto a portion of an automated push-pull mechanism 522 which could pulland/or push the spreading device 500 over the microscope slide 506 toautomate the entire spreading process (FIG. 39A, 39B). The spreadingdevice 500 may have pins or some means to attach the spreading device500 to the track 520 on the slide support element 310 or adjacentthereto or elsewhere around the slide support element 310 to move thespreading device 500 over the treatment surface 510 of the slide 506.The spreading device 500 or 500 a may be attached to the extendablepush-pull mechanism 522 via a pin 524 for example. Each reaction slidesupport element 310 and/or reaction compartment 316 of the stainingapparatus 300 (or other staining apparatus contemplated herein) can havethe ability to utilize these spreading devices to spread reagents uponthe microscope slides 506 positioned thereon. Shown in FIGS. 39A-39B isan embodiment of an automated push-pull mechanism 522 for moving thespreading device 50. When the microscope slide 506 is placed on theslide support element 310 before testing is started, the techniciancould position the spreading device 500 to the instrument and at theappropriate time a reagent 514 could be deposited on the microscopeslide 506 and the spreading device 500 could then be moved over themicroscope slide 506 to evenly apply the reagent 514 over the tissuespecimen 504. Once the entire staining process (the entire treatmentprotocol) is complete the technician could remove the microscope slide506 and spreading device 500 and discard or clean the spreading device500. In a preferred embodiment the spreading device 500 is color codedand is disposable.

In an alternate embodiment, the spreading device 500 (or 500 a)described herein can have the reagent already contained within areservoir in the spreading device 500 (or 500 a) and dispensed therefromonto the microscope slide 506. When loading the staining apparatus 300,the technician could remove a protective cover or closure device on thespreading device to expose the reagent to be applied to the microscopeslide 506. In accordance with the invention, the technician can placethe microscope slide and spreading device onto the slide support element310. Once the slide support element 310 and microscope slide 506 thereonis inside the reaction compartment 316, the reaction compartment 316 canbe depressurized or held in a vacuum. This vacuum environment can pullthe reagent out of the spreading device reservoir and onto themicroscope slide and the spreading device can then move and spread thereagent over the microscope slide as described above. In an alternateembodiment, the reaction compartment 316 can be under pressure to expelthe reagent from the spreading device reservoir. In an alternateembodiment, the spreading device is attached to an armature on the X-Y-Zpositioning device and is movable thereon, rather than on the slidesupport element or on a reagent pack.

Shown in FIGS. 40-42 is an alternate embodiment of a reagent pack of thepresent invention designated therein by the general reference numeral550. Reagent pack 550 has round configuration such as a disk shape. Thereagent pack 550 comprises a plurality of “pie-shaped” containerportions 552 each having a reagent container 554 thereon, and a centralaperture 556 through which a pin or other holding device on a reagentpack support device of the invention can engage the reagent pack 550.The reagent pack 550 operates by being rotated to an applicationposition wherein a reagent in the reagent container 554 can be expelledonto a microscope slide on a life support element of the invention.Reagent pack 550 is shown as comprising eight container portions 552 butit will be understood by a person of ordinary skill in the art that thereagent pack 550 could comprise 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12 ormore container portions 552 rather than the eight shown herein. FIG. 41Ashows reagent pack 550 taken through line 41A. In this embodiment thecontainer portion 552 comprises a “blister” or “bubble” container 554 awhich is designated to be “crushed” open. FIG. 41B shows an alternativeversion of container portion 552 taken through like 41 B of FIG. 40showing a “piston” type container 554 b wherein the reagent in thecontainer 554 b is expelled by compression of a “piston” in thecontainer 554 b which causes expulsion of the reagent therein through anaperture 560 therebelow. Represented by the reference number 558 in anembodiment of a spreading device 558 such as described elsewhere hereinwhich can be used to spread the reagent over the microscope slide. Shownin FIG. 42 is a single container portion 552 of reagent pack 550 and tabslots 568 into which the connecting tabs 566 can be inserted wherein theplurality of container portions 552 can be connected into the reagentpack 550, or could be disconnected and rearranged and reconnectedtogether. The tabs 566 are not the only connecting means to connect thecontainer portions 552 together and indeed any connecting device knownin the art for use as a connecting means could be used as long as theresulting reagent pack 550 functions in accordance with the invention.Further, the circular reagent pack may be of integral, unitaryconstruction, that is, the reagent pack may not be constructed ofseparable “pie” portions but may be constructed of a solid base.

Shown in FIGS. 43A-43B in cross-section is a staining apparatus 580which is the same and other staining apparatuses contemplated hereinexcept as described below. Staining apparatus 580 has a front wall 582and, inner space 584, and a slide support element 586 having a sealingend 590 and sealing means 596. The slide support element 586 is sized tofit into a reaction compartment 588 in a manner similar to other slidesupport elements and reaction compartments described herein except thatwhen slide support element 586 is inserted into reaction compartment 588(for sealing a microscope slide therein), the sealing end 590 of theslide support element 586 sealingly engages with a mating surface on thefront wall 582 to form a seal between the end portion 590 of the slidesupport element 586 and the front wall 582 as indicated in FIG. 43B. Anadvantage resulting from this embodiment of the invention is that aseparate door is not necessary to close the aperture in the front wall582 through which the slide support element 586 is passed. A reactionpack support device of the invention could have a similar sealing meansin an end portion thereof. Preferably the sealing end 590 is a ground orpolished glass surface as is the mating surface on the front wall 582,or it could be any similarly ground or polished surface in the materialfrom which the sealing end 590 of the slide support element 586 isconstructed. The opposite end of the slide support element 586 couldhave a similarly configured sealing end portion and in an alternateembodiment, the sealing end 590 of slide support element 586 could bedesigned to form a seal in a mating portion of an inner wall of anembodiment of the present invention wherein the staining apparatuscomprises a pressurizable common chamber such as inner wall 440 ofstaining apparatus 400 a or inner wall 440 b of staining apparatus 400b. For example in staining apparatus 400 b, the slide support element416 b could have a sealing end such as sealing end 590 which sealinglyengages wall 440 b for forming a seal therebetween, and which replacesthe door 441 b therein.

In a preferred embodiment of the present invention, a microscope slideis placed on the corresponding slide support element when it is in aposition outside of the staining apparatus. The reagent pack specificfor that particular microscope slide similarly would be placed on thecorresponding reagent pack support device (wherein the loading positionfor the reagent pack support device is inside or outside of the stainingapparatus). The reagent pack preferably would feature a bar code, OCRsymbol, machine readable symbol or code that can be read by an opticalscanner or scanners associated with the staining apparatus to determinewhat type of treatment protocol is to be performed on the correspondingmicroscope slide. Once the microscope slide has been placed on thecorresponding slide support element the technician can place theappropriate reagent pack on its reagent pack support device and press abutton nearby the slide support element or reaction compartment or frontwall of the staining apparatus or on the screen of the microprocessor tostart the treatment process. Since the lab technician knows whatparticular protocol that is required for each microscope slidepositioned, in an alternative embodiment the tech would place themicroscope slide on the slide support element corresponding thereto andplace the reagent pack on its reagent pack support device and push thereagent pack or reagent pack support device gently into the stainingapparatus. Once the reagent pack support device is moved about 0.1 to1.5 cm manually towards the staining apparatus, the reagent pack supportdevice will recognize this movement and will automatically continuemovement of the reagent pack into the staining apparatus without furtherassistance from the technician. The independently movable slide supportelement can, at this time, automatically move into the stainingapparatus when the reagent pack support device begins to automaticallymove into the staining apparatus or shortly thereafter. Once the slidesupport element and the reagent pack support device (and reagent pack)are inside the staining apparatus, the microprocessor will recognizethat a new reagent pack has been moved into the staining apparatus andthe staining apparatus will position a movable optical recognitioncharacter reader or scanner over the reagent pack's optical characterrecognition (OCR) code and that particular code with be identified as anew protocol for the microscope slide associated with that reagent pack.Preferably, there is no further assistance needed from the technicianonce the reagent pack support device is automatically moved into thestaining apparatus. The microprocessor will take over and all theinformation from the OCR code on the reagent pack will be deciphered tostart a new treatment protocol to the corresponding new microscopeslide. Since the microprocessor recognizes the OCR code present on thereagent pack, the staining process will then be carried out by all theautomated processing devices under its control. Preferably, there is noneed to have an OCR code on the microscope slide to link the slide withits reagent pack. This one step identification, of the presentinvention, is preferred versus the prior art identification of slidesand reagent container where both the slide and the reagent containerneed to have OCR code present thereon to locate and dispense the rightreagent to the right slide. This saves money and time by not placing anOCR code on the prior art microscope slide to be processed byautomation. An alternate version of identifying the reagent pack is thereagent pack can have any wireless device know in the art of recognizingwireless devices by a microprocessor. The reagent pack can have, forexample, a wireless device embedded or on the reagent pack. The reagentpack can have embedded information in the form of microchip or otherdevice to store the protocol information that can be recognized anddeciphered by the microprocessor. When the protocol and slide processingis completed, the microprocessor will alert the technician that themicroscope slide is ready to be removed from the staining apparatus.This alert can be is form of a sound and/or visual effect either nearthe particular slide support element or front wall of the stainingapparatus or on the microprocessor's screen. The notification that thetreatment protocol is completed and the slide can be removed from thestaining apparatus can be provided by any known device or devices bothaudible and/or visually known in the art of notification ofmicroprocessor controlled devices. A preferred notification is both anaudible alert, which can be of different sounds or pitches relating tothe entire process from start to removal of the microscope slide, alongwith a visual alert on the staining apparatus or on the screen of themicroprocessor.

Each slide support element of the present invention preferably has aslide support eject button, associated therewith and each reagent packsupport device preferably has a reagent support eject button associatedtherewith. Each set of reaction components preferably comprises aprotocol status indicator light or lights, “quick code” buttons, and aLCD or LED screen for visual information regarding the protocol,reagent(s), and or microscope slide.

The regent pack, strip or individually contained reagent or reagentspreferably features a bar code, OCR symbol, machine readable symbol orcode or other similar symbol that can be read by the apparatus's opticalscanner or scanners to determine what type of protocol is to beperformed on the corresponding microscope slide. The reagent pack,strip, or individually contained reagent or reagents or the microscopeslide can also have a “quick code” that corresponds to a “quick key” or“hot key” on the apparatus that can be entered into the apparatusmanually to identify the treatment protocol for a particular slide. Oncethe microscope slide is placed on its slide support the technician wouldplace the desired reagent pack on its reagent pack support device andpress a button nearby the slide support element or reaction compartmenton the front wall of the staining apparatus front panel ormicroprocessor screen to start the treatment process. Since the labtechnician knows what particular protocol that is required for eachmicroscope slide positioned on its independently moving slide supportelement, in an alternative embodiment the technician would place themicroscope slide on its slide support element and place the reagent packon its reagent pack support device and push the start button on theapparatus to initiate the automatic independent movement of the slidesupport element and reagent pack support device into the inner space ofthe staining apparatus of the apparatus. The apparatus would then readthe OCR code or symbol on the reagent pack to program the microprocessorfor that particular treatment protocol for the microscope slide on theslide support element. The microprocessor with take over and all theinformation from the OCR code on the reagent pack will be deciphered tostart a new treatment protocol to the corresponding new microscopeslide. The apparatus can also read the slide's OCR code or symbol, ifpresent, to confirm that the reagent pack selected by the techniciancorrelates to that particular microscope slide. In an alternativeembodiment, the reagent pack's OCR code can be manually scanned by awired or wireless hand held scanner for the manual programming of thetreatment protocol. The user would place the microscope slide onto aslide support element and either scan the OCR code of the reagent packprior to putting the reagent pack on the reagent support or after thereagent pack is placed onto the reagent pack support device. Theapparatus would then start the protocol by automatically moving theslide and reagent pack into the apparatus. In an alternate embodiment,the user programs the apparatus for a particular treatment protocol byentering into the apparatus or staining module a “quick code” that ispresent on the reagent pack. This “quick code” can be a number, symbol,letter, or identified by a particular color code. For example, a number“2” can be present on the reagent pack, or the letter “C” or particularcolor code like “blue”. The user would place the microscope slide on thecorresponding slide support element then place the required reagent packon the reagent pack support device and press the “quick key” on theapparatus that has the same number, letter, or color code that ispresent on the reagent pack. These quick codes can also be on themicroscope slide and/or the reagent pack. The “quick codes” are usefulwhen common or repetitive protocols are used. This speeds the time ofprogramming the apparatus for a particular repetitive protocol. Forexample, if the user has five “estrogen receptor” protocols to beanalyzed at one time, the user would place the five slides onto theircorresponding slide support elements and place the 5 reagent packs forthe “estrogen receptor” protocol onto their reagent pack supportdevices. The user would then press or activate the individual “quickcode” button or icon for that staining module that corresponds to theestrogen receptor protocol's “quick code” for each microscope slide. Forexample, the “estrogen receptor” protocol is part of a staining protocolclass known in the art as a “prognostic” test. Since all prognostictests could have the same incubation times, the “prognostic class” ofantibodies could all have the same “quick code”. The user can nowprogram all the “prognostic” protocols for each “prognostic” slide bypressing or activating the single “quick code” button to program theapparatus for a “prognostic” protocol. Seven slides for a “prognostic”panel could have, for example, seven prognostic antibodies like estrogenreceptor, progesterone receptor, Ki-67, Her-2, bcl-2, p-glycoprotein,and p53. The user would place each microscope slide on its correspondingslide support element and then place the reagent pack for that“prognostic” antibody test and then press or activate, for each of thesets of reaction components, the “quick code” button. The programmedincubation times would be the same for each module even if the antibodytest was different for each slide. Because this class of antibodiesbeing used, in this example the “prognostic” antibodies, all have thesame “quick code” on their reagent pack or slide, different prognosticreagent packs can have different prognostic antibodies present but allhave the same protocol when it relates to the incubation times for thewhole class. Another class known in the art are the “core” antibodies.These antibody protocols also have different primary antibodies in eachreagent pack, but the incubation times can be the same. The “core”antibodies can all have the same “quick code” presented on their reagentpack. They can all be a different antibody test or protocol only theyall have the same incubation times for each step. An example of thistype of class of antibodies tests can have the letter “A” on theirreagent pack. The user would then press or activate the “A” buttonassociated with the staining module and the test would start. The “quickcode” buttons can be pre-set at the factory or can be user manipulateddepending on the user preference. Each “quick code” button can beprogrammed with a different protocol incubation time or any othervariant relating to protocol method and stored for future use with that“quick code” button. It would be known that any variant to this methodcan be used. Whether the slide is placed first and the reagent pack isplaced second or vice versa is anticipated. Also whether or not theslide support element or reagent pack support device are moved into orare outside of the apparatus before operation is contemplated. Any stepof moving of the slide support element or reagent pack support device,either semi-automatically or completely automatically is contemplated.The steps of placing the microscope slide on the slide support element,the placement of the reagent pack on the reagent pack support device,automatic scanning of the OCR code, manual scanning of the OCR code, andpressing or activation of the “quick code” buttons can all be used inany combination, method and or sequence. Each individual slide supportelement can automatically move outside the staining apparatus to place amicroscope slide thereon or for removal of the microscope slide when atest is complete by pushing the slide support eject button on theapparatus. The slide support element and reagent pack support device canalso be semi-automatically moved outside or inside the stainingapparatus by manually moving the slide support element or reagent packsupport device about 0.1 to 1.5 cm thereby activating their automaticmovement mode. Once the slide support and/or the reagent support ismanually moved about 0.1 to 1.5 cm inwards towards the stainingapparatus or moved towards the front wall thereof, the slide supportelement and/or the reagent pack support device would recognize thismanual movement and the apparatus will take over by automatically movingthe slide support element and/or reagent pack support device into or outof the staining apparatus. This movement is operationally similar to themode of operation of a computer CD-ROM drive door or drawer of DVDmachine drawer. In an alternate embodiment the slide support element andthe reagent pack support device can move totally automatically andindependently by pushing a button on the staining apparatus to initiatesaid movement. A button for insertion or ejection of the slide supportelement and reagent pack support device can be present for each set ofreaction components on the front panel (front wall) of each stainingmodule. The insertion/ejection button can be a single button for movingboth or may comprise separate buttons for each movement.

As explained elsewhere herein, the slide support element and thereaction compartment can be made out of glass with a polished sealmatingly sealing the inner surface of the reaction compartment and theouter surface of the slide support element. For example, a glass syringecommercially available from Popper and Sons can be modified for thisembodiment. A Perfecktum™ glass hypodermic syringe (cat no: 5159, 50 ccsyringe) or equivalent could be modified to produce a glass slidesupport element (constructed from the inner barrel or plunger of thesyringe) and a glass reaction compartment (constructed from the outerbarrel of the syringe). The sealing means is the polished glass betweenthe inner barrel (the slide support element) and the outer barrel of thesyringe (the reaction compartment). This polished glass mating sealbetween the slide support element and the reaction compartment enablesthe slide support element to be easily moved into and out of, androtated within, the reaction compartment. For example, the slide supportelement can be tilted, spun, or otherwise rotated within the reactioncompartment as well as be moved laterally forward and backwards while inthe reaction compartment. The advantage of this design is that the slidesupport element, inside the reaction compartment is able to moveforward, backwards, and in a circular motion (rotated) while forming andmaintaining a pressure tight seal inside the reaction compartment formedby the polished glass seal between the slide support element andreaction compartment. The circular, rotational, motion is ideal to“spin” the slide support element to cause removal of a reagent or washsolution from the slide by centrifugal force. The reagent is “spun” awayfrom the microscope slide and drained from the reaction compartment andis then ready for the next reagent or can be “spin dried” prior toremove of the microscope slide from the slide support element. The slidesupport element, because of the polished glass seal, is very easilymoved within the reaction compartment. For example, in one version, asimple twist of the slide support element can cause the slide supportelement to make several revolutions within the reaction compartment evenif the reaction compartment is under positive (or negative) pressuresthat exceed (or are below) atmospheric pressure. The microscope slidecan be in any position to be washed by a wash reagent dispenser andthen, if necessary blown off by a gas pressure dispenser, with the slideat any angle on the slide support element. The home position for themicroscope slide is when the upper surface of the slide faces upward(the “12:00 o'clock” position or 0°). The slide could be washed at the12:00 o'clock position, the 3:00 o'clock position (90° from homeposition), the 6:00 position (180° from home position), 9:00 positiono'clock (270° position) or any degree position between the homeposition(0°) and 360° from home position. The preferred positions forwashing the slide would be between the 0° position (home position) and180° (6:00 position). Slide processing devices can be positionedanywhere around the slide support elements to dispenses reagents, gas,or other processing device proposes at any angle the microscope slide ispositioned on the movable slide support element. For example, thestaining reagents (antibodies, molecular probes, biological stains,detection reagents, pre-treatment reagents, antigen retrieval solutions,or other reagent or solution described herein) could be dispensed to themicroscope slide from above the slide support element in the homeposition (“12 o'clock” or “0 degree” position) and then the microscopeslide could be rinsed at the “6:00 o'clock” position (180° position) bya rinse wash reagent dispenser and then spun dried to remove the washreagent and then drained from the reaction compartment.

The electrical connections to each individual heating element or otherelectrical device on or in the slide support element or reactioncompartment can be controlled by wireless connections, Bluetooth®connections, impedance connections, or any other type of wirelessconnection to enable the free movement of the slide support element andreaction compartment in any direction or speed or speed of movementthereof. For example, the individual heating element that is part of theslide support element can be connected to the microprocessor wirelesslyby those connections known in the art of connecting electrical deviceswirelessly. This wireless connection of the individual heating elementcan thus be maintained when the slide support element or reactioncompartment are in motion, for example, this enables maintenance of theheating current to the individual heating element when the slide supportelement is spinning while removing reagents by centrifugal force.

The reaction compartments and/or the slide support elements of theinvention optionally are disposable. The disposable slide supportelement can be constructed of plastic or polymers that can support amicroscope slide and be able to withstand the temperature and pressurerequirements of the present invention. Pressures of 25-30 psig andtemperatures of 100-160° C., for example, are possible with modernplastics, thermoplastics, and polymers. In one embodiment, thedisposable slide support element is constructed without a heatingelement, rather the heating element used to heat the reagent to theabove mentioned temperatures is placed within the walls of the reactioncompartment rather than in the slide support element. A disposablereaction compartment is also contemplated. The disposable reactioncompartment can be constructed using the same materials as saiddisposable slide support element. Heating elements for heating themicroscope slide could be, for example, heaters that can be presentoutside of the disposable reaction compartment or disposable slidesupport element. In one embodiment, the heating element can be tubularand can contain, in its center, a disposable reaction compartment in atubular shape. The walls of such a tubular heater could heat the tubularreaction compartment and thus heat the reagent associated with the slidesupport element. After a microscope slide has been treated thedisposable slide support element, and/or the disposable reactioncompartment can be removed from the apparatus and discarded. A newdisposable reaction compartment can then be placed into the tubularheater and/or a new disposable slide support element can be placed inthe staining apparatus for use. All the motions and controls of thepresent invention can be utilized with this embodiment of disposablereaction compartments and disposable slide support elements.

In an alternate embodiment of the invention, a plurality of slides areprocessed (either separately in individual reaction compartments orwithin a common vessel) by applying a reagent or solution to the slideand pressurizing the vessel above atmospheric pressure to levels asdiscussed elsewhere herein, wherein the biological specimens,biochemicals, or other biological entity on the slide is not subjectedto additional heating.

As described elsewhere herein, preferably the slide support element,reaction compartment, reagent pack, reagent pack support device,dispensing element, ports, conduits, mixing jets, pressurizing means,cooling means, aspiration devices, drainage ports, heating devices, andreagent conduits are independently operable and independently movable.

The in situ antigen recovery and staining apparatus of the presentinvention preferably has as one component a device for reading ordetecting an optical character or code which identifies a reagent packor reagent pack component such as a tile or container.

As noted above, “stirring” of the reagent on the microscope slide can beperformed by applying a gas stream onto the microscope slide to impart acircular motion of the reagent on the microscope slide. Alternatively,the mixing of the regents can be from sonic, ultrasonic, and orvibratory waves passing through the reagent causing agitation of thereagent on the microscope slide. These waves cause a physical movementof the static fluid state of the reagent. The movement of the reagentcauses the liquid phase to move or mix the reagent on the microscopeslide and increase exposure of the reagent to the biological specimenthereby increasing the reaction of the reagent with the biologicalspecimen on the microscope slide. Further, these mixing processes can beuseful in agitating a rinse reagent to effectively remove the unboundreagent from the biological specimen thus producing a stained biologicalspecimen with low or no background staining. These mixing processes canbe on the surface of the liquid and/or the center of the liquid and/orthe bottom of the liquid to agitate or mix the reagent.

These mixing processes act to decrease the time necessary to process abiological specimen present on the microscope slide. The liquid reagentmust come in intimate contact with the biological specimen for thebiological reaction to take place. The staining of biological specimenwith biological stains, monoclonal antibodies, polyclonal antibodies,molecular RNA and DNA probes, immunoreagents, detection reagents,chromogens and counterstains and other such reagents, also referred toherein as “reagent elements” utilize heat and time to passively producethe required reaction of these reagents with the biological specimenherein known as “biological elements”. One embodiment of the presentinvention utilizes a magnetic field to “direct” these “reagent elements”to their respective targets associated with the biological specimen. Amagnetic field can be generated from below, above, or adjacent themicroscope slide with an electromagnet which is capable of reversing itspolarity. This electromagnet of the present invention can impart apowerful magnetic field to align a “regent element” and draw it towardsthe biological element present in the biological specimen. Theelectromagnet and method of use herein is contemplated. Reagents likemonoclonal and polyclonal antibodies are known and used routinely in thedetection of biological antigens. These testing antibodies are uniquelyattracted by their corresponding antigens present in the biologicalspecimen. This embodiment of the present invention utilizeselectromagnets below the biological specimen to “align” and “pull” theseantibodies toward the biological specimen therefore decreasing theprocessing time of these antibodies versus simply passively placing theantibodies on the biological specimen in the prior art methods. In thisembodiment of the present invention an electromagnet or permanent magnetis placed in or adjacent the slide support element that supports themicroscope slide and its biological specimen thereon. The reagent isplaced on the upper side of the microscope slide and the electromagnetis energized with an appropriate polarity required to produce a magneticfield between the top side of the liquid reagent and the topside of themicroscope slide with the biological specimen between the top side ofthe liquid reagent and the top side of the microscope slide. Thismagnetic field in this embodiment of the present invention pulls theregents elements (e.g., antibody, probe, stain) toward the top side ofthe microscope slide. As the magnetic field pulls and directs thereagent elements toward the top side of the microscope slide, thereagent elements pass through or closely thereto the biological specimenand effectively and efficiently physically attach to or associate withtheir respective biological elements. If the desired reagent element hasa net positive charge on its active binding site, the electromagnet ormagnet would impart a net negative charge to attract and pull thereagent element toward the biological element, for example. If thereagent element has a net negative charge on its active binding site,then the electromagnet or magnet would impart a positive magnetic fieldto attract the reagent element toward its biological element, forexample. The entire processing protocol, relating to positive andnegative field generation, by the electromagnet or magnet, is controlledby the microprocessor and is directed according to the protocolselected. The liquid reagent can be of any type and composition known inthe art of staining microscope sides. The composition of the diluentspresent in the prior art compositions for diluting the active reagentare known and can be use with the present invention. The preferredembodiment of the present invention utilizes the magnetic field alone tohave a direct effect on the reagent element or elements present in theliquid testing reagents of the prior art. No alteration or additionalchemicals, other than the known standard liquid reagents diluents orstandard buffers, are necessary for the method of the present inventionswherein an electromagnet or magnets is used to cause a direct pull andmoving effect on the reaction element in relation to the biologicalelements. The present invention may use only the magnetic fieldgenerated by the electromagnet or magnet to act specifically on thereagent elements present in known diluting buffers, regardless of thetype of diluting buffer used to make a working solution of liquidreagent. The present invention method is not dependent on the solvent orbuffer being used to dilute the reagent elements for testing. Thepresent invention preferably relies only on the net charge of thereagent element present in the known and widely accepted diluentsbuffers along with a very strong magnet either an electromagnet,permanent, superconducting, and or resistive magnet. The Tesla rating ofthe magnet can be 0.000001 Tesla to 60 Tesla. One Tesla equals 10,000Gauss. Preferably the Gauss rating can be 1 to 20,000 Gauss. Commondiluents buffers are phosphate buffer saline and Tris® based diluents,with or without detergents present, and a preservative. The reagentelements need only to have a net positive or negative charge to be usedwith the embodiments of the present invention. The electromagnetic forceimparted on the reagent element in combination with the high positivepressure or negative pressure in the reaction compartment, produces anenvironment that substantially decreases the amount of time needed toreact a biological specimen with a reagent element. In a furtherembodiment of the present invention, a magnetic field is used to causemovement of reagent element about a biological element. For example, thebuffer used or diluents to used dilute reagent elements can have iron oriron-like element present therein to be acted on by the magnets. Theiron micro or nano particles are present to act as a motile device formixing or agitation of the reagent elements about the biologicalelements. Micro or nano iron particles that are coated with an inertplastic or polymer can be used to mix, agitate, or move the reagentelements in their respective diluents. The micro or nano iron particlesare moved by the magnet current supplied by the magnets present aroundthe biological elements. Other magnetically moving particles are alsocontemplated. The reagent elements themselves can have attached tothemselves iron or iron-like micro particles to move, mix, or attractreagents to the biological elements. Examples of these particles thatcan be acted on by magnets are particle like colloidal gold orbiochemicals to which the colloidal gold is attached. Colloid gold isroutinely conjugated to antibodies and nuclei acid probes. The colloidalgold can be seen in electron microscopy and light microscopy whendeveloped by a silver enhancing protocol. The particle size of the goldparticle is 1-10 nm but can be smaller or larger. The smaller the sizeof the gold particle is important because the extra weight of the goldparticle attached to the reagent element is proportional to the movementand attachment sites of the reagent element to the biological elements.Examples of the present invention using magnets to mix, agitate, or movereagent elements are described. The colloidal gold particle is attachedto a reagent element and the magnetic attraction of the gold particle isused to pull the reagent element towards its biological element. Thismovement reduces the time necessary for incubation times of the reagentelement to “find” its biological elements. The magnetic conjugatedreagent element being pulled toward the biological elements by themagnet, along with the pressure of the present invention leads tosubstantially reduced time of incubation. The micro particles can be anyferro containing particle (Fe) or other metal particles that can bemoved by a magnet are know and contemplated. The particle can be of thesize less than 1×10⁻¹⁰, 1×10⁻⁹, 1×10⁻⁸, 1×10⁻⁷, 1×10⁻⁶, and up to 1×10⁻⁵meters.

The nano or micro-particles can be coated with a plastic, polymer,coating to help in the stability of the particles in solutions. Thecoating can be Teflon®, fluropolymer, plastic, or ceramic. The particlecan be by itself in the reagent diluents or attached to the reagentelement. The particle can be soluble, at least partial soluble, orcolloidal in the diluents solution. If the particle is not attached to areagent element is would be used to mix or agitate the surroundingsolution. If the particle is attached to the reagent element it can befor mixing, agitating, or moving the reagent element. In an alternativeembodiment the diluents can have present a electrolyte present toproduce a net charge of the reagent elements present and to further theeffects of the magnet on the reagent elements.

Magnets that can be used in the present invention are contemplated asbeing permanent magnets, superconducting magnets, resistive magnets. Thepreferred embodiment is the use of a permanent magnet that has hightemperature stability for the use in the present inventions chamberedhigh pressure and high temperature conditions. High temperature stablepermanent magnets such as described in U.S. Pat. No. 6,451,132 which ishereby incorporated by reference in its entirety can be used. Thesemagnets are represented by the general formula RE(Co_(w) Fe_(v) Cu_(x)T_(y))_(z), where RE is a rare earth metal selected from the groupconsisting of Sm, Gd, Pr, Nd, Dy, Ce, Ho, Er, La, Y, Th, and mixturesthereof and T represents a transition metal(s) selected from the groupconsisting of Zr, Hf, Ti, Mn, Cr, Nb, Mo, W, V, Ni, Ta, and mixturesthereof. These high temp permanent magnets can be subjected totemperatures exceeding 700° C. These high temperature permanent magnetscan be incorporated into the heating element of the slide support oradjacent thereof. These magnets can be a single high temperaturepermanent magnet that is constructed along with a conductive heatingelement producing the slide support base or pad where the slide touchesthe slide support. The magnet can have the heating source sandwichedbetween two magnets or the heating source can be above the magnet orbelow the magnet. The magnet itself can be the slide support element ora portion thereof which is able to be heated by a conductive typeheating source or plate. The heating element can be on top, between, orbelow the magnetic slide support element. The magnet and heater can beseparated from the slide by a glass slide support base, for example. Theentire slide support element can be constructed out of high temperatureglass like Pyrex® wherein the slide base, where the microscope slide ispositioned and rests on, or in the slide support element during thestaining protocol. Underneath such glass slide support base is themagnet and heater which is outside the slide support element in relationto the slide on the glass base. The heater alone or the heater andmagnet can be sealed within the glass of the slide support element. Thisembodiment wherein the heater and or heater and magnet is sealed withinthe glass slide support insulates or protects the heater or heater andmagnet from corrosive chemicals during staining. This insulation awayfrom the inner space of the slide support element and or slide base bythe heater is a preferred embodiment whether there is a magnetassociated with the heater or not. With the embodiment of the slidesupport element and reaction compartment being constructed of glass orother material, which is describe elsewhere, preferably produces anenvironment of all glass surfaces touching the microscope slide andareas adjacent to the microscope slide as a preferred embodiment of thepresent invention. Since in this embodiment there is no non-glassexposed part of the slide support element when the slide support elementis inside the glass reaction compartment any chemical that is corrosiveor incompatible with metals or plastic can now be used. Any of theprocessing components can be insulated by the glass slide support orglass reaction compartment. The glass slide support element can have,molded within the glass, tabs or notches or other means present to alignand hold the microscope slide to the glass microscope slide base holdingthe microscope slide on the glass slide support element. The magneticfield and the conductive heater can act on the microscope slide andspecimen through the glass base of the slide support element with theadvantage of the heater and magnet not being exposed to the chemicals ofthe inner compartment of the slide support element. Glass is a goodconductor of heat and the magnetic field. Other magnets like Neodymiummagnets are a type of permanent magnet that can have the ability toretain their magnetic properties even under very high temperatureconditions. Most permanent magnets lose their magnetic properties whenthey are exposed to high heat conditions. The type of permanent magnetcontemplated for the present invention has the grade of N42SH the “SH”grade of Neodymium permanent magnets can be used in temperatures over150° C. Standard “N” grade permanents magnets have a maximum operatingtemperature of 80° C. A “SH” grade Neodymium permanent magnet with thedimensions of 2 inches long by 1 inch wide by one eighth inch has aGauss rating of 3095 for its surface field strength. It also has a Brmaxof 13,200 Gauss and a BHmax of 42 MGOe. This magnet could be justunderneath the slide support element heating plate (heating element) forexample or it may be incorporated into the heating plate or be part ofthe heating plate. The magnet can be automatically independently movableto change the polarity of the current in relation to the biologicalelements or reagent elements.

These permanent magnets can be of any shape or size to be used in thepresent invention. The mentioned magnets can be in the slide supportelement, the heating element or rod in the reaction compartment, or inreaction compartment walls. The magnet can be outside the slide supportelement and outside the reaction compartment and be adjacent to oneslide support element and one reaction compartment or the magnet ormagnets can be in the walls of the reaction compartment or positionedinside or outside the walls of the instruments case. Further, theautomated instrument described above can be inside a bore of a magnet.The instrument describe above can be made of glass and of non-ferrousmaterial to have all the advantages of being used under the strength ofvery powerful magnets.

As explained previously, the staining apparatus contemplated herein canuse a reconfigurable, rearrangeable or configurable individualizedreagent dispensing device such as a pack, strip, or single “dose” whichcomprises a carrier or holder for holding one or more individualizedregent dispensers wherein the regent dispensers is movable fordispensing a desired sequence of reagents (or just a single reagent) toa biological specimen on the slide. Each such pack is individualized tobe in association with only a single biological specimen on a slide. Thepack and/or the reagent container thereon can be disposable or reusable.As noted above, examples of such packs shown in U.S. PublishedApplication 2006/0275861 which is entirely incorporated herein byreference.

Various embodiments of the processes of the present invention include,but are not limited to, (1) application of a reagent to a slide usingthe present apparatus, and heating the slide, with or without a step ofpressurizing the reaction compartment, (2) filling the base cavity witha reagent or solution such that it immerses the slide, pre-pressurizingthe reaction compartment, then heating the slide and reagent solution inthe base cavity, (3) filling the base cavity with a reagent or solution,then heating the slide and reagent or solution, withoutpre-pressurization before the heating step, or (4) placing a liquid inthe bottom of the base cavity without the liquid directly touching theslide, then heating the liquid in the base cavity to cause vaporformation which pressurizes the reaction compartment and secondarilyheats the slide and reagent therein (the slide also may optionally beheated by the slide heater).

Other aspects of the present invention are shown and described in U.S.Provisional Application Nos. 60/142,789; 60/684,047; 60/689,386 and60/730,744, U.S. Patent Application Publication No. 2006/0275861, and WO2006/127852, the entirety of each of which is hereby expresslyincorporated herein by reference.

The heating element or plate of the slide support elements can beslightly smaller than the width of a microscope slide to facilitateremove of the slide from the heating plate. The width of the heatingplate can be 1-6 millimeters, for example, less than a microscope slidewidth. A standard microscope slide is about 25 mm in width. The heatingplate can be 23 mm, for example, in width to facilitate removal of themicroscope slide off the heating plate.

The slide support element can have an ejection means such as a movablepin or lever underneath the microscope slide to push up a portion of theslide to facilitate removal of the microscope slide from the heatingplate. These ejection means can be underneath one or more corners of themicroscope slide for example. This movement can facilitate the cleaningunderneath the microscope slide, removal of the microscope slide, orcooling of the microscope slide by moving the slide away from theheating plate.

The heating plate can have holes present for vacuum or pressure to beapplied to the bottom of the microscope slide. Pressure exerted fromthese holes can push up the microscope slide to help remove the slidefrom the heating plate. The holes can also be used to help cleanresidual reagent that may be trapped underneath the slide. The processof using a rinsing liquid and the use of the vacuum or pressure holes inthe heating plate provides a method of cleaning and drying the undersideof the microscope slide.

The staining apparatus can have automatically leveling devices, reactioncomponents such as slide supports and reaction compartments, pins, pegs,feet, or level sensors that are under the control of the microprocessor.When the apparatus is turned on the microprocessor will determine if theentire apparatus and or each reaction component is level. If it is notlevel or needs to be adjusted the leveling devices (stepper motors,pneumatic, electromechanical devices) in each leveling device, slidesupport, reaction compartment, pins, pegs, feet are moved in or out tolevel the entire apparatus or each reaction component. This isespecially important when using the field models since they are movedmore frequently. The main microprocessor can determine if the entireapparatus or each staining module is level each time the apparatus isturned on or a “level” icon can be available on the mastermicroprocessor to level or check the levels at any time during aprotocol.

The staining apparatus can produce a blast of air inside the reactioncompartment of agitate a reagent or liquid therein to produce anemulsion.

Mixing a reagent on the microscope slide can be by at least one gassource blowing across the slide to stir the reagent. Mixing can occur byblowing at least one gas jet over the reagent and subsequently movingthe slide support in at least one direction to agitate or mix a reagentor rinse a slide. Mixing is very efficient because the present inventionutilizes agitated rinse or kinetic rinsing to dislodge unbound reagentsfrom the biological specimen or the microscope slide. The kineticmovement can be by gas, physical movement of the slide, vibrations,agitation, ultrasound, etc. Kinetic movement can be for mixing orrinsing.

There can be a separate individual camera present on the outside frontwall of each staining apparatus of the apparatus to see the label end ofthe microscope side or reagent pack information more clearly or increasethe visual size of the microscope labeled end or reagent packinformation. The camera can, for example, inversely project its image toimprove viewing of the label end of the microscope slide for betteridentification of the name of the stain desired.

The reagent pack can have a RFID (radio frequency identification) tag ordevice for the apparatus to automatically identify the reagent pack andprotocol program.

The apparatus can use non- refrigerated reagent packs for field and labuse to reduce necessary refrigeration space.

The reagent container, capsule, or vial can line up to the reagentconduit on the reaction compartment or window, or over the microscopeslide and a vacuum can pull the reagent out of the capsule or vialwithout using the dispensing element to push the reagent out. The vacuumpulls the reagent out and the reagent drips onto the microscope slide.

There can be a plurality of movable reagent conduit lines each having amagnetic end to connect the reagent conduit line to the metal reagentconduit positioned on the reaction compartment. One of the heads on theat least one X-Y-Z positioning device can have a plurality of thesemovable reagents lines with magnetic couple ends to service one or aplurality of reaction compartment simultaneously with a remote reagentfrom a reagent container or bulk reagent bottle.

The X-Y-Z positioning device can be constructed so as to be able to pickup different types of spreading devices from a supply station and usethem on the microscope slide to spread reagents. When the reagent isspread across the slide, the dispensing head, carrying the spreadingdevice, can move to an ejection area to eject the used spreading deviceand can return to the supply station to pick up a new spreading device.

The X-Y-Z positioning device can be of any type known in the art ofdispensing reagents. There can be one or a plurality of X-Y-Zpositioning devices that can move independently to reagent supplystations or spreading device supply stations to pick up and dispensereagents from a remote source inside the staining apparatus or outsidethe staining apparatus.

A wet, recently floated, tissue section on a microscope side can beplaced onto a slide support element and is moved into an individualreaction compartment or common pressurization chamber to apply pressureto the tissue section to further flatten out the section to themicroscope slide before, during, or after the heat plate is turned on tomelt the paraffin and securely attach the tissue or biological specimento the microscope slide.

The microscope slide once stained can be coverslipped by a dry filmadhesive glass coverslip by applying a solvent to the slide then tiltingthe slide support element at an angle to the coverslip dispenser andthen the coverslip is touched at one edge to the microscope side and theslide support is moved back to horizontal placing the coverslip on theslide. The heating plate is turned on to dry the coverslip prior toremoval of the slide for examination under a microscope.

The present invention contemplates that the microscope slides andreagents used herein can be heated by magnetic induction. Thisembodiment would be in the place of wired heating elements in theindividual reaction compartment and individual slide support element.The reaction compartment and or slide support element would have metalassociated therewith for magnetic induction heating.

Magnetic Induction heating is the process of heating an electricallyconducting object, like a metal, by electromagnetic induction.Electromagnetic induction heating is the production of voltage across aconductor situated in a changing magnetic field or a conductor movingthrough a stationary magnetic field (Faraday's Law). This changingmagnetic field generates eddy currents within the metal and theresistance leads to Joule heating of the metal. This type of heater isknown, for example, in the art of cooking ranges and cook top surfaces(Waring Pro SB-30, Pro ICT100, Waring Products 314 Ella T. GrassoAvenue, Torrington, Conn. 06790). An induction heater (for heating areagent on or around the biological specimen or just the biologicalspecimen) consists of an electromagnet, through which a high-frequencyalternating current (AC) is passed. Commercial power line frequency isacceptable to induce the primary inductor or electromagnet. Heat mayalso be generated by magnetic hysteresis losses in materials that havesignificant relative permeability. The frequency of AC used depends onthe object size, material type, coupling (between the work coil and theobject to be heated) and the penetration depth. Magnetic induction worksbest with cast iron, steel, stainless steel, ferrite based metal(s) andany coated metal of these types. The cast iron, steel, stainless steel,ferrite based metal(s) can be coated or intergraded with glass, ceramicor enamels, for example to have excellent anti- corrosive properties.Any coating known in the art of metal coating that can be heated can beuse and are contemplated. Copper to some degree can be used. Magneticinduction heating doesn't heat non-metal objects. The primary inductor(electromagnet) would be positioned around the metal slide supportelement heating plate, or any other metal associated with a slidesupport, reaction compartment, common camber, reagent support, reagentcontainers, reagent conduits, etc. The metal slide support element ormetal heat plate, or magnetic induction inducible heating material, forexample, is heated by a commercial power line frequency (current)induced in it by a primary inductor (electromagnet). This type ofheating of any metal present in the staining apparatus that is requiredto be heated to transfer (conduction heating) the heat to a reagent orjust the biological specimen is advantageous in the present invention.Just the metal in the reaction compartment and slide support elementwould get hot to heat the reagent. The individual reaction compartmentcan be constructed of metal, metal and glass, metal and ceramic, ormetal and a plastic polymer for use with a magnetic induction heatingdevice. The individual slide support element can be constructed ofmetal, metal and glass, metal and ceramic, or metal and a plasticpolymer for use with a magnetic induction heating device. Since thepresent invention has independently moving processing components (i.e.—independently moving slide supports, independently moving reactioncompartments, independently moving reagent supports, etc.) this methodof heating doesn't require hard electrical wiring to each heater orheaters. This use of magnetic induction to heat reagents or thebiological specimen or both, reduces the clutter and cost of hard wiringeach heater(s) of the present invention. Each reaction module orstaining module can have at least one separate independently workingmagnetic inductor to heat an electromagnetic inducible metal that canthen conductively transfer its heat to a reagent or biological specimenfor a particular heat requiring protocol. There may also be more thanone magnetic inductor for heating more than one metal source of thereaction module or staining module. The heated plate(s) or heated metalthat is heated by Joule heating is extremely fast, controllable, andefficient. The heated metal plates or heated metal structures can beregulated in the range of less than 1° C. to exceeding 1000° C. Morepreferable the temperature regulation can be in the range of 20° C. to180° C., depending on the heating requiring protocol. Any processingdevice can be constructed of an electromagnetic inducible metal and canhave any shape. Shapes made of an electromagnetic inducible metal liketubes, plates, pins, ducts, dispensers, supports, of all types of shapesand construction are known and are contemplated. The processing devicesof the present invention would be constructed mostly of non-metalmaterials and only the heating areas being constructed of an induciblematerial like metal. The microprocessor can regulate the temperature ofany electromagnetically-inducible metal by adjusting the voltage orcurrent to the at least one primary inductor (electromagnet) thereforeregulating the electromagnetic inducible metal(s) (i.e., slide supportelement, heat plate, reaction compartment heated wall(s), reagent stripsupport heater, reagent containers heater, etc.) temperature associatedwith each component of the staining apparatus. It is known that any andall type of heating method along with magnetic induction heating iscontemplated and any combination of these types of heaters (i.e. —infrared, conductive, convection, radiant, foil, kapton, conductive inks,magnetic induction, microwaves, etc.) can be used in each slide supportelement or reaction compartment. The electromagnetically induciblemetal(s) can be quickly cooled once the primary inductor is turn off,because it is not necessary to wait for the heating means to cool downas well. When the electromagnetic induction is turned off the heat stopsgenerating at the inducible metal site and the cooling process startsimmediately without having to wait for the heating source (i.e.,electromagnetism) to cool down along with the heated inducible metal.Just the inducible metal is cooled alone. This is in stark contrast forthe cooling method of a conventional conduction heat source whichrequires the cooling of the conduction heat source in lockstep with itsheated plate. The reaction compartment can be made entirely of glass orceramics as to not be heated by the magnetic induction heating device.The inside of the reaction compartment can be engineered to be themagnetic induction heating device that heats the slide support elementmetal heat plate or the entire slide support element if it wasconstructed of metal. The advantage to this is the outside of thereaction compartment can remain cool to the touch and only the slidesupport element or slide support would be heated by the magneticinduction device to heat the reagent present on or associated with themicroscope side. A user can place a bench unit, field unit, or smallscale version of the staining apparatus (e.g., comprising 5-15 reactioncompartments) near their microtome or processing table duringpreparation of a microscope slide once the tissues is floated onto themicroscope slide the user can press the individual slide supportelement's eject/insert button and the individual slide support elementinside the staining apparatus would then automatically move out of thestaining apparatus and the user could then place the wet microscopeslide onto the individual slide support element. The user could thenpress the appropriate button on the staining apparatus to cause theelectromagnet to induce the individual slide support element metal platedirectly under only the microscope slide to start heating the microscopeslide with biological specimen attached. The slide support element metalplate would be heated causing heating of the microscope slide thereonwithout heating the remainder of the slide support element because it isconstructed from a non-metal material like glass, for example. If theuser would accidently touch the slide support element he or she wouldnot feel the heat because only the heating plate of the slide supportelement and microscope slide thereon are being heated and the majorityof the slide support elements mass (i.e., glass slide support) is notheated. The user can then let the slide support element stay outside thestaining apparatus or move the slide support element into the stainingapparatus by slightly pushing in on the slide support element toactivate the automated movement of the slide support element into thestaining apparatus. The user can alternatively press the eject/insertbutton again to automatically move the slide support element into thestaining apparatus without pushing in on the slide support element. Thismovement is similar to a CD-ROM drawer or door on a personal computerand is described in detail elsewhere in this application. Once all ofthe microscope slides are placed on their individual slide supportelements the user would move all the slide support elements into thestaining apparatus either by pushing each individual eject/insert buttonfor each slide support element or press the appropriate icon to move allthe open slide support elements into the staining apparatus at the sametime. There are icons and buttons present on the staining apparatus tomove just one slide support element out of the staining apparatus andinto the staining apparatus or move all the slide support elementstogether out of the staining apparatus or into the staining apparatus.An alternate embodiment of the present invention using magneticinduction heating is the use of a disposable individual slide supportelement and or a disposable individual reaction compartment or both thathas at least one area being metal or an inducible metal or material thatcan be heated by magnet induction is contemplated. A further example isthe use of a metal pan or inducible material in the cavity of the slidesupport element or the metal pan or inducible material in the head spaceof the reaction compartment. The magnetic induction heating device wouldthen only heat the metal pan or inducible material in the slide supportelement, therefore heating D.I. water, for example, in the metal pan toproduce steam that would pressurize the reaction compartment and heatthe reagent on or associated with the biological specimen on themicroscope slide. The method of magnetic induction heating contemplatedherein is preferred because it can be controlled precisely depending onthe amount of heat required and the amount of steam being generated toproduce the desired level of pressurization without the necessity ofreleasing of the pressure being produced by steam generation to controlpressure level. The magnetic field can be adjusted to regulate the heattemperature of the metal pan therefore increasing or decreasing thepressure contained in the reaction compartment for pressure regulation.Any combination of metal and non-metal in the construction of theindividual reaction compartment or individual slide support element iscontemplated. A magnetic induction heating device can be in or aroundthe individual reaction compartment and/or in or around the individualslide support element. Magnetic induction can be used as long as thereis metal or an inducible material either in the reaction compartmentand/or metal present in the slide support element that can be heated bya magnetic induction heating device. The pressurizable common chambercan also employ magnetic induction to heat the walls of thepressurizable common chamber and or the metal slide support elements orareas requiring heating by magnetic induction of a metal or induciblematerial inside the pressurizable common chamber.

The staining apparatus can be relatively small , having just 5-20 setsof reaction components for example. This compact “point of use” stainingapparatus can be positioned at the microtome or cryostat. A user canplace wet microscope slides with their newly floated tissue sectionattached or frozen tissue attached onto the staining apparatus at thepoint of microtomy. Once the slides have been placed onto the stainingapparatus, the apparatus can be moved to an area for staining the slidesor just left near the microtome or cryostat to start the stainingprocess. The automated leveling feature (described elsewhere in thisapplication), of the present invention, can “level” the stainingapparatus prior to staining or treatment initiation. The user needs onlythe reagent pack for each particular slide protocol to be placed into oronto the reagent pack support device and start the protocol. The entirereagent protocol, including rinses and application of a coverslippingmountant, can be provided by the reagent pack with no need for bulkfluid sources if desired. The entire protocol from start to finish ispreferably supplied from the reagent pack. If the apparatus requiresbulk fluid sources, the apparatus can have attached bulk fluids incontainers that can be small and quickly refillable without stopping thestaining apparatus because the bulk fluid containers can be linkedtogether in a series or parallel for quick removal, filling, or disposalof bulk reagents and bulk waste.

The staining apparatus in one embodiment is adapted for pressurizedpre-treatment only. It is constructed so as to perform only HighPressure Epitope Retrieval (HiPer™) pretreatments without furtherstaining the slide. This HiPer™ apparatus can perform “Heat InducedEpitope Retrieval” [HIER] and or High Pressure Epitope Retrieval(HiPer™) pretreatment protocols. This embodiment is useful in particularwhen labs have an existing manual or automated staining platform orsystem that needs the added benefit of quick and efficient hightemperature pre-treatment protocols prior to placing slides onto theirexisting automated or manual staining systems. The HiPer™ apparatus canuse reagent packs for different types of heat induced epitope retrievalsolutions or bulk fluid containers for use with the ports in eachreaction module. The HiPer™ apparatus can move individual slides intoand out of a pressurizable common chamber without leakage of thepressure contained in the pressurizable common chamber. The HiPer™apparatus features Independent Access™, the mechanics of which aredescribed elsewhere in this application.

The HiPer™ apparatus can also be adapted to move a plurality of slideson a single slide support device into and out of a pressurizable commonchamber for a pre-treatment under pressurization, prior to furtherstaining. A plurality of slides movable on a common support can be movedinto and out of the pressurizable common chamber. The plurality ofslides is moved into the inner space of the common chamber; a reagentcan be dispensed onto each individual microscope slide eitherindependently or simultaneously. This apparatus can use reagent packs ordispense reagents from a bulk reagent solution container by ports suchas dispenser elements described elsewhere in this application. Thepressurizable common chamber is closed and is subjected to pressure andheat to treat the biological specimen on the microscope slide. Theheating means and pressurization means are explained elsewhere in thisapplication. The reagent on or associated with the biological specimenis preferable on only the microscope slide.

The staining apparatus of the invention, in any embodiment describedherein, can have a hand held or stationary scanner like IRISPen™ Express6 (I.R.I.S. Group s.a. 10 rue du Bosquet, B-1348, Louvain la Neuve,Belgium) or any scanner or digitizer that can “scan” the entiremicroscope slide before, during and or after the biological specimen hasbeen processed. Any scanner or digitizer known in the art can be used.This scanner or scanners provides information to the exact location orthe position of the biological specimen (i.e., tissues section(s)) onthe microscope slide in relation to the frosted, Colormark™,Colorfrost™, or otherwise labeled end of the microscope slide. Thescanner can also use or store the information provided on the labeledend. The scanner can scan before, during, or after the slide is stainedto store information to give the user the digital account of the entirestaining protocol that can be stored in memory of the microprocessor andbe retrieved at a later date for evaluation. The stored information canbe for any OCR code or codes on the slide's labeled end along with thedigital image of the biological specimen before, during, and after thecompleted processing or staining. The scanner may also be inside thestaining apparatus and is movable inside the staining apparatus such asdescribed previously in regard to the X-Y-Z processing device. Furthereach set of reaction components can have an independently moving scannerspecific to only one set of reaction components. The scanner(s) can bestationary and the slide support element is movable to provide thescanning motion. The scanner can be inside the staining apparatus oroutside the staining apparatus or both. There is at least one scannerpresent for the staining apparatus to capture digital images of thebiological specimen on the microscope slide and the labeled end of themicroscope slide, an example being, the tissue section can be scannedand the staining apparatus detects where the biological specimen ispositioned relative to the labeled end of the microscope slide. Thestaining apparatus can now more effectively and efficiently dispense ortreat only the area of the microscope slide the biological specimenoccupies. The location, area used by the biological specimen, andbiological specimen(s) information (i.e., size, area, pieces oftissue(s) present, cells, agglutination patterns, color, texture, inkingcolors for margin identification, etc.) along with the informationcollected from any OCR code, machine readable code(s), letters, numbers,symbols, written information, etc. present on the labeled end of themicroscope slide can be compiled, calculated, arranged, digitallystored, and retrieved for later analysis.

The reagent(s) used with the spreading device described in regard toFIGS. 37-39B can have an additive to help spread or give substance orbody to the reagent being spread by the spreading device. Thickeningagents like Xanthan gum, glycols, thickeners, polyols, with or withoutdetergents like Brij, Tween, Igepal, ionic and non-ionic detergents canbe present in any reagent to be spread by the spreading device.

TREATMENT PROTOCOL EXAMPLES Example 1

-   -   (1) Place microscope slide on slide support element and enclose        within reaction compartment;    -   (2) Add antigen recovery buffer;    -   (3) Set slide heater at 130° C.;    -   (4) Pressure regulator set at 23 psig (259.9 kPa);    -   (5) Antigen recovery buffer reaches 125° C.;    -   (6) Incubate at 125° C. for 10 minutes;    -   (7) Turn off heater and turn on air or liquid cooling system;    -   (8) Cool 5 minutes; and    -   (9) Rinse with buffer and proceed with staining protocol.

Example 2

-   -   (1) Place microscope slide on support element;    -   (2) Enclose microscope slide within individual reaction        compartment;    -   (3) Dispense 1-2 ml of antigen retrieval reagent onto microscope        slide;    -   (4) Close all external ports;    -   (5) Open pressure port to pre-pressurize reaction compartment to        about 25 psig (273.7 kPa);    -   (6) Turn on heat plate to reach about 120° C. on slide;    -   (7) Set pressure regulator to maintain 120° C. temperature by        regulating the reaction compartment's pressure;    -   (8) Reagent reaches a temp of 120° C.;    -   (9) Heating is maintained for 30 minutes at about 120° C.;    -   (10) Turn off heater and turn on air or liquid cooling system;    -   (11) Cool 5-10 minutes;    -   (12) Release pressure to atmospheric pressure;    -   (13) Cool antigen retrieval reagent;    -   (14) Rinse slide with PBS wash buffer; and    -   (15) Proceed with staining protocol.

Example 3

Three mis of antigen recovery buffer present in reaction compartment canbe heated to a particular reaction temperature at a particular pressure,including for example: 100° C. @ 8 psig (156.6 kPa), 106° C. @ 10 psig(170.3 kPa), 110° C. @ 12 psig (184.0 kPa), 115° C. @ 15 psig (204.7kPa), 120° C. @ 16 psig (211.6 kPa), 125° C. @ 23 psig (259.9 kPa), or130° C. @ 30 psig (308.1 kPa), 140° C. @ 40 :retrieval buffer after a 60minutes treatment time.

Example 4

Ambient temperature with pressure staining protocol:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent pack or other dispensing element;    -   4) Pressurize the chamber with a separate gas to desired        pressure (50-100 psig: 446-790.6 kPa);    -   5) Incubate the reagent for a desired time (10-120 minutes);    -   6) Depressurize the chamber by opening the waste port;    -   7) Rinse slide of reagent by rinsing and/or tilting and rinsing        the slide;    -   8) Repeat steps 3-7 until all reagents are dispensed for a        particular protocol and for a desired time.

Example 5

High temperature Antigen Retrieval protocol with pre-pressurization:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent pack or other dispending element        onto the microscope slide;    -   4) Pressurize the chamber with a separate gas to desired        pressure (15-30 psig: 204.7-308.1 kPa);    -   5) Turn on at least one heating element (i.e., slide heater,        chamber heater, cavity heater) and heat to 125° C.;    -   6) Pressure is maintained at 15-20 psig (204.7-239.2 kPa) by the        pressure release valve or heating modulation (i.e., hearing        elements turning off and on);    -   7) Incubate reagent at 125° C. for 10-30 minutes;    -   8) Turn heaters off and turn on cooling ducts (liquid or air)        until reagent drops below 50° C.;    -   9) Depressurize the chamber sending condensation and pressure        out the waste port;    -   10) Rinse slide of reagent by rinsing and/or tilting and rinsing        the slide;    -   11) Dispense regent and incubate with or without pressure and/or        with or without heat for a desired time;    -   12) Repeat steps 9-10 until all reagents are dispensed.

Example 6

High temperature Antigen Retrieval protocol without pre-pressurization:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent pack or other dispending element        and fill up the chamber with reagent by totally immersing the        entire slide in reagent (i.e., antigen retrieval reagent);    -   4) Turn on at least one heating element (i.e., slide heater,        chamber heater, cavity heater) and heat to 125° C.;    -   5) Pressure is produced by the reagent boiling;    -   6) Pressure is maintained at 25 psig (273.7 kPa) by the pressure        release valve or heating modulation (i.e., heating elements        turning off and on);    -   7) Reagent is incubated at a temperature of 125° C. for 10-30        minutes;    -   8) Turn heaters off and turn on cooling ducts (liquid or air)        until reagent drops below 50° C.;    -   9) Depressurize the chamber sending condensation, reagent, and        pressure out the waste port;    -   10) Rinse slide or reagent by rinsing and/or tilting and rinsing        the slide;    -   11) Dispense reagent and incubate with or without pressure        and/or with or without heat for a desired time;    -   12) Repeat steps 10-11 until all reagents are dispensed.

Example 7

High temperature Antigen Retrieval protocol—cavity produces steam tomaintain high heat conditions with pressurization:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent pack or other dispending element        onto the microscope slide;    -   4) Add deionized (D.I.) water, or other liquid reagent to the        cavity below the slide (deionized water not contacting the        microscope slide);    -   5) Turn on slide heating element and cavity heaters and heat to        125° C.;    -   6) Pressure is produced by the deionized water boiling in the        cavity and producing steam to heat the reagent on the microscope        slide;    -   7) Pressure is maintained at 25 psig (273.7 kPa) by the pressure        release valve or heating modulation (i.e., heating elements        turning off and on);    -   8) Reagent is incubated at a temperature of 125° C. for 10-60        minutes;    -   9) Turn heaters off and turn on cooling ducts (liquid or air)        until reagent drops below 50° C.;    -   10) Depressurize the chamber sending condensation, deionized        water and pressure out the water port;    -   11) Rinse slide of reagent by rinsing and/or tilting and rinsing        the slide;    -   12) Dispense reagent and incubate with or without pressure        and/or with or without heat for a desired time;    -   13) Repeat steps 10-11 until all reagents are dispensed.

Example 8

Using the individual sets of reaction components with hand held orstationary reagent OCR code reader:

-   -   1) Push or press eject/load button on individual reaction module        (i.e., set of reagent components) front panel (near the reaction        compartment opening or individual eject/load icon on computer        screen for the chosen reaction module).    -   2) The individual slide support element ejects outside or moves        out of the staining apparatus.    -   3) Place the microscope slide onto the individual slide support        element (e.g., onto the hotplate).    -   4) The individual digital camera projects the labeled end of the        slide on the microprocessor screen for better viewing of written        information on the labeled end of the microscope slide.    -   5) Chose the correct reagent pack and hand scan the reagent pack        OCR or code.    -   6) The microprocessor loads the correct protocol and information        for that particular reagent pack.    -   7) The microprocessor opens the reagent pack door and the        reagent pack support device ejects or moves outside the staining        apparatus.    -   8) Place the reagent pack on the reagent pack support device and        press the start button at the individual reaction module front        panel or press the start icon on the microprocessor screen        related to that particular reaction module.    -   9) Both the independently moving slide support element and        independently moving reagent pack support device automatically        move into the staining apparatus independently.    -   10) Protocol initiates.    -   11) After the protocol is completed, the individual reaction        module will have both a sound and visual alert to the finish        protocol.    -   12) Press the finished button on the individual reaction module        front panel or icon on the microprocessor screen.    -   13) The finished slide is ejected out of the staining apparatus        for removal from the independently moving slide support element.    -   14) The used reagent pack is then eject or removed from the        staining apparatus and is discarded.    -   15) The slide support element and reagent pack support device is        then moved back into the staining apparatus.

Example 9

Using the individual reaction modules with automated reagent OCR reader:

-   -   1) Push or press eject/load button on individual reaction module        front panel (near the reaction compartment opening or individual        eject/load icon on computer screen for the chosen reaction        module).    -   2) The individual slide support element ejects outside or moves        out of the staining apparatus.    -   3) Place the microscope slide onto the individual slide support        element (e.g., onto the hotplate).    -   4) The individual digital camera projects the labeled end of the        slide on the microprocessor screen for better viewing of written        information on the labeled end of the microscope slide.    -   5) Push or press eject/load button of the reagent pack support        device.    -   6) The microprocessor opens the reagent pack door and the        reagent pack support device ejects or moves outside the staining        apparatus.    -   7) Place the reagent pack on the reagent pack support device and        press the start button at the individual reaction module front        panel or press the start icon on the microprocessor screen        related to that particular reaction module.    -   8) Both the independently moving slide support element and        independently moving reagent pack support device automatically        move into the staining apparatus independently.    -   9) The scanner inside the staining apparatus reads the OCR code        or code on the reagent pack.    -   10) The program is now loaded along with the information of the        reagent pack.    -   11) Protocol automatically initiates.    -   12) After the protocol is completed, the individual reaction        module will have both a sound and visual alert to the finish        protocol.    -   13) Press the finished button on the individual reaction module        front panel or icon on the microprocessor screen.    -   14) The finished slide is ejected out of the staining apparatus        for removal from the independently moving slide support element.    -   15) The used reagent pack is then eject or removed from the        staining apparatus and is discarded.    -   16) The slide support element and reagent pack support device is        then moved back into the staining apparatus.

Example 10

Using the individual reaction modules with a “quick code”:

-   -   1) Push or press eject/load button on individual reaction module        front panel (near the reaction compartment opening or individual        eject/load icon on computer screen for the chosen reaction        module).    -   2) The individual slide support element ejects outside or moves        out of the staining apparatus.    -   3) Place the microscope slide onto the individual slide support        element (e.g., onto the hotplate).    -   4) The individual digital camera projects the labeled end of the        slide on the microprocessor screen for better viewing of written        information on the labeled end of the microscope slide.    -   5) Push or press the reagent pack “quick code” on the individual        reaction module front panel or icon on the microprocessor        screen.    -   6) The program is now loaded along with the information of the        reagent pack.    -   7) The microprocessor opens the reagent pack door and the        reagent pack support device ejects or moves outside the staining        apparatus.    -   8) Place the reagent pack on the reagent pack support device and        press the start button at the individual reaction module front        panel or press the start icon on the microprocessor screen        related to that particular reaction module.    -   9) Both the independently moving slide support element and        independently moving reagent pack support device automatically        move into the staining apparatus independently.    -   10) Protocol automatically initiates.    -   11) After the protocol is completed, the individual reaction        module will have both a sound and visual alert to the finish        protocol.    -   12) Press the finished button on the individual reaction module        front panel or icon on the microprocessor screen.    -   13) The finished slide is ejected out of the staining apparatus        for removal from the independently moving slide support element.    -   14) The used reagent pack is then eject or removed from the        staining apparatus and is discarded.    -   15) The slide support element and reagent pack support device is        then moved back into the staining apparatus.

In summary, the invention in one embodiment contemplates an in situantigen recovery and staining apparatus, comprising a plurality ofindependently operable reaction compartments having an inner space, aslide support element able to support a microscope slide in the reactioncompartment, the slide support element positionable within or adjacentthe inner space of the reaction compartment for sealing the microscopeslide therein wherein the reaction compartment is pressurizable (oroptionally depressurizable) to maintain an internal pressure whichexceeds (or is below) atmospheric pressure, and a dispensing element(e.g., reagent pack, port, or plunger) for dispensing a reagent onto themicroscope slide while the reaction compartment is pressurized (oralternatively, not pressurized), and may further comprise a heatingelement for heating the microscope slide upon the slide support element.

The staining apparatus may further comprise a reagent pack supportdevice for supporting a reagent pack having one or more reagentcontainers which contain or is able to contain a reagent therein,wherein the reagent pack support device supports the reagent pack in aposition external to the reaction compartment and/or the slide supportelement, and the dispensing element may be adapted to engage the reagentcontainer of the reagent pack thereby causing the reagent to bedelivered from the reagent container into the inner space of thereaction compartment and onto the microscope slide or directly only themicroscope slide disposed directly on the slide support element insideor outside of the reaction compartment.

Preferably, each of the reaction compartments of the staining apparatusis individually and independently pressurizable (or, optionally,depressurizable) and each of the heating elements is individually andindependently operable and heatable.

In the staining apparatus, the reaction compartment may be pressurizablebefore, during, or after the heating element heats the microscope slide,the heating element may be a component of the slide support element andmay be positionable directly beneath the microscope slide, the reactioncompartment may have a cylindrical, tubular shape wherein the slidesupport element has a cylindrical shape, or the reaction compartment mayhave a rectangular shape, such that the slide support element has arectangular shape.

In the in situ antigen recovery and staining apparatus, each slidesupport element is preferably independently movable in relation to eachother slide support element, each reagent pack is independently movablein relation to each other reagent pack, each reaction compartment isindependently movable in relation to each other reaction compartment,and each dispensing element (plunger, etc.) is independently movable inrelation to each other dispensing element. The reaction compartment ispreferably pressurizable to maintain a pressure above atmosphericpressure, such as 0 to 350 psig (101.3-2514 kPa), to a pressure of 1 to100 psig (108.2-790.6 kPa), to a pressure of 5 to 50 psig (135.8-446.0kPa), or to a pressure of 10 to 40 psig (170.3-377.0 kPa), or isdepressurizable to maintain a pressure below atmospheric pressure to alevel as low as 100 kPa to 10 kPa to 1 kPa to 100 Pa to 10 Pa to 1 Pa to0.1 Pa.

In the staining apparatus, the reagent disposed onto or about themicroscope slide may be heated, for example, to a temperature of 25° C.to 37° C., 37° C. to 56° C., 56° C. to 85° C., 85° C. to 100° C., 100°C. to 125° C., 125° C. to 135° C., 135° C. to 150° C., 150° C. to 175°C., 175° C. to 200° C., 200° C. to 225° C., 225° C. to 250° C., 250° C.to 275° C., 275° C. to 300° C., 300° C. to 325° C., or 325° C. to 350°C. The reaction compartment, when present, may be stationary or movable,and each reagent pack support device associated therewith may bestationary or movable. When the slide support element is stationary, thereaction compartment may be movable, and the reagent pack support devicemay be stationary or movable. When the slide support element of thereaction module is movable or stationary, and the reaction compartmentis movable, and the reagent pack support device is movable, the reactioncompartment may be movable independently of the reagent pack supportdevice. Further, the reagent pack support device may be movable ineither a forward or reverse direction to carry the reagent pack whenloaded thereon in either a forward or reverse direction, and when thereagent pack support device is stationary, the reagent pack may bemovable in either a forward or reverse direction when loaded thereon.The reagent pack support device and the reaction compartment may beconnected to each other, or not connected. Each reaction compartment maycomprise at least one of (1) an air duct for pressurizing the reactioncompartment or causing mixing of the reagent on the slide, (2) a coolingduct for enhancing the rate of cooling of the heating element afterheating, (3) a supply port for delivering a liquid to the slide supportelement, and (4) a drainage duct for removing reagents supplied to themicroscope slide. The staining apparatus may comprise a separate reagentconduit for enabling delivery of reagent from each reagent pack into thecorresponding reaction compartment, a heating device disposed about thereagent conduit for heating the reagent delivered therethrough, aheating device for heating the reaction compartment, and a heatingdevice in the reagent pack support device for heating the reagent packor portions thereof.

The slide support element of the apparatus may have a cavity in aposition below the microscope slide for containing a quantity ofsolution and the cavity may have a cavity heater for heating thesolution within the cavity. The dispensing element may be operableindependently of the reagent pack support device (e.g., as a componentof the X-Y-Z positioning device, and the dispensing element preferablyfunctions to cause expulsion of reagent from a reagent container of thereagent strip and/or to dispense a reagent or solution from a reagent orsolution source remote from the reagent pack such as from the remotereagent source. The slide support element may receive reagent from thereagent pack or reagent or solution from a remote source when the slidesupport element is disposed inside or outside of the reactioncompartment. The dispensing element is preferably able to apply suction,or is able to apply liquid, air, or gas under pressure. The slidesupport element may be enclosable within the reaction compartment bymoving the slide support element into the reaction compartment or bymoving the reaction compartment about the slide support element. Theslide support element may be tiltable to allow drainage of reagent orsolution from the microscope slide. The plurality of sets of reactioncomponents can be assembled into at least one chamber to form a stainingapparatus. Each slide support element, reagent pack support device,dispensing element, and reaction compartment of the staining apparatusis preferably separately replaceable or exchangeable, and preferably hasmeans for controlling or releasing pressure from or regulating pressurewithin the reaction compartment.

The present invention also contemplates a reconfigurable reagentdispensing pack, comprising a plurality of reagent module, each reagentmodule comprising a tile and a reagent container secured thereto, eachreagent module preferably adapted to be attachable to and detachablefrom an adjacent reagent module such that once the plurality of reagentmodules are attached together in a first sequence, one or more of thereagent modules can be detached and reattached to reconfigure theplurality of reagent modules in a second sequence different from thefirst sequence. The reconfigurable reagent dispensing pack may have aconnecting link for connecting adjacent reagent modules, and an injectorfor enabling a reagent within the reagent container to be dispensed fromthe reagent container, and the reagent container may be removable fromthe tile in one embodiment. Further, at least one of the reagentcontainers contains a reagent selected from the group consisting ofantigen retrieval reagents, RNA and DNA probes, citrate buffer, EDTA,TRIS, PBS, with or without surfactants or detergents like SDS, Tween,Brij, ionic and non-ionic detergents, and silicone additives, rinsebuffers, immunohistochemical reagents, histochemical reagents, in-situhybridization reagents, PCR reagents, coverslipping reagents, siliconeoils, mineral oils, detection reagents and processing reagents, liquidreagents, reconstituted dry reagents, biological reagents and aqueousand non-aqueous reagents, and deparaffinizing compositions of water withone or more silicone surfactants or silicone additives.

Alternatively, the reconfigurable reagent dispensing pack may comprise abase, having a plurality of container platforms, and a plurality ofreagent containers, with each container platform having a reagentcontainer secured thereto, wherein each reagent container is adapted tobe attachable to and detachable from the container platform such thatonce the plurality of reagent containers are attached together in afirst sequence, one or more of the reagent containers can be detachedand reattached to a different container platform to reconfigure theplurality of reagent containers in a second sequence different from thefirst sequence, thereby forming a reconfigured reagent dispensing pack.The reagent container may be positioned upon a tile which is detachablefrom the base. The reagent container or container platform may furthercomprise an injector for enabling a reagent within the reagent containerto be dispensed from the reagent container.

Alternatively, the reconfigurable reagent dispensing pack may comprise aplurality of reagent modules, each reagent module comprising a tile anda reagent container secured thereto, wherein the tiles are initiallyconstructed in a unitary, integral configuration and each tile isadapted to be attachable to and detachable from an adjacent tile suchthat the reagent modules are connected in a first sequence, and whereinwhen one or more of the tiles is detached, the one or more tiles can bereattached to reconfigure the plurality of reagent modules in a secondsequence different from the first sequence, and may further comprise aconnecting link for re-connecting tiles of adjacent reagent modules. Thereagent module may further comprise an injector for enabling a reagentwithin the reagent container to be dispensed from the reagent container,and the reagent container may be removable from the tile.

In another embodiment, the present invention contemplates a method oftreating a microscope slide, comprising: providing a plurality ofindependently operable reaction compartments each having an inner space,a plurality of slide support elements each able to support at least onemicroscope slide in a horizontal position, the slide support elementpositionable within or adjacent the inner space of the reactioncompartment for sealing the microscope slide therein, and a dispensingelement for dispensing a reagent into the reaction compartment, thendisposing the microscope slide onto the slide support element,positioning the microscope slide within the reaction compartment,pressurizing the reaction compartment to maintain an internal pressurewhich exceeds atmospheric pressure, and actuating the dispensing elementto cause the reagent to be delivered into the reaction compartment whilethe reaction compartment is pressurized and wherein the reagent isdelivered at a pressure which exceeds the pressure within the reactioncompartment, and optionally heating the microscope slide and reagentwithin the reaction compartment.

Preferably the invention comprises a method of treating a microscopeslide, comprising, providing a plurality of independently operablereaction compartments each having an inner space, a plurality of slidesupport elements able to support a microscope slide, the slide supportelement positionable within or adjacent the inner space of the reactioncompartment for sealing the microscope slide therein, a heating elementfor heating the microscope slide, a reagent pack support device forsupporting a reagent pack having a plurality of reagent containers eachof which contains or is able to contain a reagent therein, wherein thereagent pack support device supports the reagent pack in a positionexternal to and adjacent the reaction compartment, and a dispensingelement for engaging the reagent container thereby causing the reagentto be delivered from the reagent container onto the microscope slide,and wherein each of the reaction compartments of the plurality ofreaction modules is individually and independently pressurizable (or,optionally, depressurizable) and wherein each of the heating elements ofthe slide support elements is individually and independently heatable.The microscope slide disposed on the slide support element, the slidesupport element and microscope slide thereon is then positioned withinthe reaction compartment, the heating element is activated to heat theslide, the reaction compartment is then pressurized to maintain aninternal pressure which exceeds atmospheric pressure.

In the method, the step of pressurizing (or depressurizing) the reactioncompartment may occur before, during, or after the heating of themicroscope slide by the heating element. The reaction compartment mayhave a cylindrical, tubular shape for enhancing pressure distributionwithin the reaction compartment. The slide support element of eachreaction module may be moved independently in relation to each otherslide support element, each reagent pack may be moved independently inrelation to each other reagent pack, and each dispensing element may bemoved independently in relation to each other dispensing element. Thereaction compartment may be pressurized to a pressure of above 0 to 350psig (101.3-2514 kPa), to a pressure of 1 to 100 psig (108.2-790.6 kPa),to a pressure of 5 to 50 psig (135.8-446.0 kPa), or to a pressure of 10to 40 psig (170.3-377.0 kPa). The reaction compartment may bedepressurized to maintain a pressure below atmospheric pressure to alevel as low as 100 kPa to 10 kPa to 1 kPa to 100 Pa to 10 Pa to 1 Pa to0.1 Pa. The reagent disposed onto or about the microscope slide may beheated to a temperature of 25° C. to 37° C., 37° C. to 56° C., 56° C. to85° C., 85° C. to 100° C., 100° C. to 125° C., 125° C. to 135° C., 135°C. to 150° C., 150° C. to 175° C., 175° C. to 200° C., 200° C. to 225°C., 225° C. to 250° C., 250° C. to 275° C., 275° C. to 300° C., 300° C.to 325° C., to 325° C. to 350° C. The step of positioning the slidesupport element may comprise moving the slide support element into thereaction compartment while the reaction compartment is stationary, orthe step of positioning the slide support element may comprise movingthe slide support element and moving the reaction compartment. Thereagent pack may be positioned in a dispensing position by moving thereagent pack support device thereby moving the reagent pack to thedispensing position, or by moving the reagent pack while the reagentpack support device is stationary. The method may comprise moving theslide support element of the reaction module, moving the reactioncompartment is movable, and moving the reagent pack support device,wherein the reaction compartment is movable independently of the reagentpack support device.

While the invention has been described herein in connection with certainembodiments so that aspects thereof may be more fully understood andappreciated, it is not intended that the invention be limited to theseparticular embodiments. On the contrary, it is intended that allalternatives, modifications and equivalents are included within thescope of the invention as defined by the appended claims. Thus theexamples and embodiments described herein, which include preferredembodiments, will serve to illustrate the practice of this invention, itbeing understood that the particulars shown are by way of example andfor purposes of illustrative discussion of preferred embodiments of thepresent invention only and are presented in the cause of providing whatis believed to be the most useful and readily understood description ofprocedures as well as of the principles and conceptual aspects of theinvention.

Changes may be made in the construction and the operation of the variouscomponents, elements and assemblies described herein or in the steps orthe sequence of steps of the methods described herein without departingfrom the spirit and scope of the invention as defined in the followingclaims.

1. A microscope slide staining system, comprising: a chamber having aninner space; a plurality of independently movable slide support elementspositioned in the inner space of the chamber, each of the slide supportelements configured to support a microscope slide; a spreading devicesupply station containing a plurality of spreading devices, each of thespreading devices being positionable in association with at least one ofthe microscope slides supported on the slide support elements in a waythat the spreading device defines a gap between the spreading device andthe microscope slide when the microscope slide is positioned on theslide support element and in a way that each of the spreading devices isindependently movable relative to the other spreading devices to spreadat least one reagent on at least a portion of the microscope slide; atleast one positioning device operable in a way to pick up and carry atleast one of the spreading devices from the spreading device supplystation to one of the slide support elements so that the at least onespreading device is associated with the microscope slide supported onthe slide support element; and a plurality of heating elements such thatat least one of the heating elements is associated with one of the slidesupport elements to heat the at least one reagent on the microscopeslide.
 2. The microscope slide staining system of claim 1, wherein eachof the spreading devices engages side edges of the microscope slide whenthe microscope slide is positioned on the slide support element.
 3. Themicroscope slide staining system of claim 1, wherein each of thespreading devices slides longitudinally along the microscope slide whenthe microscope slide is positioned on the slide support element.
 4. Themicroscope slide staining system of claim 1, wherein the gap is suchthat the reagent is caused to spread across the gap by capillary actionupon the spreading device contacting the reagent.
 5. The microscopeslide staining system of claim 1, wherein each of the spreading deviceshas a reservoir in which at least one reagent is stored in a way thatthe reagent is dischargeable from the reservoir onto the microscopeslide when the microscope slide is positioned on the slide supportelement.
 6. The microscope slide staining system of claim 1, furthercomprising a plurality of reagent packs with at least one reagentcontainer on each of the reagent packs containing at least one reagent,the reagent packs supported above the slide support elements such thatthe reagent contained in the reagent containers can be expelled from thereagent containers onto a respective one of the microscope slides whenthe microscope slide is positioned on the slide support element.
 7. Themicroscope slide staining system of claim 6, wherein each of the reagentpacks has a plurality of reagent containers with each reagent containercontaining at least one reagent, the reagent packs being rotatable suchthat the reagents contained in the reagent containers can be selectivelyexpelled from the respective reagent containers onto the respective oneof the microscope slides when the microscope slide is positioned on theslide support element.
 8. The microscope slide staining system of claim1, wherein the positioning device is operable in a way to pick up andcarry the spreading device from the slide support element to an ejectionarea.